Controlled electrophoresis method

ABSTRACT

An electrophoresis apparatus is generally disclosed for sequentially analyzing a single sample or multiple samples having one or more analytes in high or low concentrations. The apparatus comprises a relatively large-bore transport capillary which intersects with a plurality of small-bore separation capillaries and includes a valve system. Analyte concentrators, having antibody-specific (or related affinity) chemistries, are stationed at the respective intersections of the transport capillary and separation capillaries to bind one or more analytes of interest. The apparatus allows the performance of two or more dimensions for the optimal separation of analytes. The apparatus may also include a plurality of valves surrounding each of the analyte concentrators to localize each of the concentrators to improve the binding of one or more analytes of interest.

RELATED APPLICATION

This application is a divisional of copending U.S. patent applicationSer. No. 10/728,499, filed Dec. 5, 2003, published as 2005-0155861 onJul. 21, 2005, and which claims priority to provisional application No.60/518,186 filed on Nov. 7, 2003. The entire contents of both of theseapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the analysis of chemical andbiological materials and, more particularly, to an improvedelectrophoresis apparatus which simultaneously performs multipleanalyses on a plurality of analytes.

2. General Background and State of the Art

Electrophoresis is a known technique for separating and characterizingconstituent and/or biological molecules, or analytes, present in simpleand complex matrices undergoing analysis. Candidate sample compoundsinclude drugs, proteins, nucleic acids, peptides, metabolites,biopolymers and other substances which exist in simple and complexforms.

Conventional systems are based on interchangeable cartridges which housea thin capillary tube equipped with an optical viewing window thatcooperates with a detector. Sample solutions and other necessary fluidsare placed in vials (cups) positioned beneath inlet and outlet ends ofthe capillary tube by means of a rotatable table.

When high voltage is applied to a capillary filled with an appropriatesolution and/or matrix, molecular components migrate through the tube atdifferent rates and physically separate them. The direction of migrationis biased toward an electrode with a charge opposite to that of themolecules under investigation. As the molecules pass the viewing window,they are monitored by a UV and/or other detector which transmits anabsorbance and/or appropriate signal to a recorder. The absorbanceand/or appropriate values are plotted as peaks which supply qualitativeand quantitative analytical information in the form ofelectropherograms.

Electrophoresis separation relies on the different migration of chargedparticles in an electric field. Migration speed is primarily influencedby the charge on a particle which, in turn, is determined by the pH ofthe buffer medium. Electric field strength, molecular size and shape ofthe analyte, temperature of the system, and other parameters alsoinfluence migration behavior.

Electrophoresis is a family of related techniques that perform highefficiency separations of large and small molecules. As one embodimentof this science, capillary electrophoresis is effective for obtainingrapid and highly efficient separations in excess of one-hundred-thousandplates/meter. Because it is a non-destructive technique, capillaryelectrophoresis preserves scarce physical samples and reducesconsumption of reagents. A fused silica (quartz) capillary, with aninner bore diameter ranging from about 5 microns to about 200 micronsand a length ranging from about 10 centimeters to about 100 centimeters,is filled with an electrically conductive fluid, or backgroundelectrolyte, which is most often a buffer. Since the column volume isonly about 0.5 to about 30 microliters, the sample introduction volumeis usually measured in nanoliters, picoliters and femtoliters (ideally2% of the total volume of the column). As consequence, the masssensitivity of the technique is quite high.

Improved instrumentation and buffer-specific chemistries now yieldaccurate peak migrations and precise area counts for separated analytes.But, capillary electrophoresis is still limited by concentrationsensitivity.

To overcome this deficiency, a series of solid-phase microextractiondevices have been developed for selective and non-selective molecularconsolidation. These devices, which are used on-line with a capillarytube, are commonly known as analyte concentrators containing affinityprobes to bind target compounds. Typical embodiments are described inU.S. Pat. No. 5,202,010 which is incorporated by reference in thisdisclosure. Other relevant teachings are provided by U.S. Pat. No.5,741,639 which discloses the use of molecular recognition elements; andU.S. Pat. No. 5,800,692 which discloses the use of a pre-separationmembrane for concentrating a sample.

Even with the advent of analyte concentrators, there is still a need toimprove the sensitivity levels for the samples that exist insub-nanomolar quantities. This deficit is particularly acute in theclinical environment where early detection of a single molecule may beessential for the identification of a life-threatening disease.

Known capillary electrophoresis instruments are also limited bylow-throughput, i.e., the number of samples that can be analyzed in aspecified period of time. U.S. Pat. No. 5,045,172, which is incorporatedby reference, describes an automated, capillary-based system withincreased analytical speed. The '172 patent represents a significantimprovement over the prior art. But, throughput is still relatively lowbecause the instrument uses only one capillary which performs singlesample analyses in approximately 30 minutes.

U.S. Pat. No. 5,413,686 recognizes the need for a multi-functionalanalyzer using an array of capillary tubes. Like other disclosures ofsimilar import, the 86 patent focuses on samples having relatively highconcentrations. There is no appreciation of the loadability andsensitivity necessary for analyzing diluted samples, or samples presentat low concentrations in a variety of liquids or fluids.

Based on these deficiencies, there exists an art-recognized need for anelectrophoresis instrument having higher loadability, betterdetectability of constituent analytes, faster throughput andmulti-functional capability for analyzing a plurality of components in asingle sample and/or a plurality of samples with high and lowconcentrations of components using a variety of chromophores, detectorsand/or pre-concentration devices.

OBJECTS OF THE INVENTION

Accordingly, it is a general object of the present invention to providean improved electrophoresis apparatus having at least one transportcapillary, at least one separation capillary and an analyte concentratorpositioned there between.

It is another object of the present invention to provide anelectrophoresis apparatus having greater operating efficiency,detectability and throughput.

An additional object of the present invention is to provide auser-friendly, sample preparation step which is designed to eliminateunwanted analytes that occupy binding sites and contaminate the innerwalls of capillaries or channels.

A further object of the present invention is to provide anelectrophoresis apparatus that can analyze multiple samples having asingle constituent, or multiple constituents of a single sample, ormultiple constituents of multiple samples.

It is a further object of the present invention to provide anelectrophoresis apparatus which uses more than one analyte concentratorto sequentially bind more than one analyte in a single complex matrix,or in multiple matrices of simple or complex configuration.

It is yet another object of the present invention to provide anelectrophoresis apparatus having enhanced loadability and sensitivitywhich is capable of analyzing samples present in a wide range ofconcentrations, including those found at low concentrations in dilutedliquids or fluids with simple or complex matrices.

It is a further object of the present invention to provide anelectrophoresis apparatus that delivers high-throughput for screeningand analyzing a wide variety of samples in multiple application areas,utilizing a single or multiple dimension separation principle or mode.

Another object of the present invention is to provide an electrophoresisapparatus which uses more than one separation method to sequentiallypermit binding to, and elution from, an analyte concentrator to effectthe separation of one or more analytes.

It is another object of the present invention to provide an automated,miniaturized desk-top electrophoresis apparatus for bioanalysis andother applications.

Additional objects of the present invention will be apparent to thoseskilled in the relevant art.

SUMMARY OF THE INVENTION

In one aspect of the invention, a sample including a number of analytesof interest is passed through a relatively large-bore transportcapillary orthogonal to a plurality of smaller-bore separationcapillaries. An analyte concentrator is positioned at each intersectionof the transport capillary and separation capillaries.

After the sample has been passed through each of the analyteconcentrators, and after the analytes of importance are captured by eachconcentrator matrix, a selected buffer is applied to each analyteconcentrator to free the system of salts and other non-relevantcomponents. For example, a typical buffered solution is sodiumtetraborate having a pH in the range of 7.0 to 9.0. The bound analytesare then eluted from each concentrator matrix in a sequentiallytime-controlled fashion using an aliquot or plug of an optimal elutingsolution. The process continues until each of the analytes has beenremoved from the concentrator matrices and passed through the detectorby high resolution electrophoresis migration. To increase thesensitivity of the analytes, an additional analyte concentratorcontaining a chromophoric reagent may be placed in one or more of theseparation capillaries to react with the analyte present in thatcapillary. Alternatively, the eluting solution may contain achromophoric reagent allowing decoupling and derivatization to occursimultaneously. The derivatized analytes can then be isolated in theseparation capillary.

To separate and analyze multiple samples with the electrophoresisapparatus of the invention, individual separation capillaries areprovided, each of which contains an analyte concentrator that enrichesthe analytes present in dilute solutions of low concentration orenriches the analytes present at low concentrations in solutions ofsimple or complex matrices containing constituent components at a widerange of concentrations. Multiple elutions are carried out in a mannersimilar to that performed when analyzing a single sample. Effectiveresults can also be achieved using solutions that contain an appropriateeluting chemical and a chromophoric reagent to simultaneously elute thetargeted analyte and enhance sensitivity. As with a single-sampleanalyzer, an extra analyte concentrator may be placed in one or more ofthe separation capillaries to allow on-line derivatization of analytes,prior and/or after separation conditions, to achieve even furtherenhancement of concentration sensitivity. In addition, an extra analyteconcentrator may be placed in one or more of the separation capillariesto permit chemical and/or biochemical reactions, such as the on-linecleavage of proteins to generate peptides.

An analyte concentrator may also be used to quantify enzymatic productsgenerated by the action of one or more pharmacological agents during aspecific enzyme reaction. Furthermore, the use of an analyteconcentrator coupled to a different mode of electrophoresis can be usedto differentiate structurally related substances present in biologicalfluids or tissue specimens. For example, the identification andcharacterization of natural proteins from artificially-made proteins orother chemicals in serum.

All reactions described above can be performed in an apparatuscontaining a format that includes either capillaries or channels. Inaddition, the migration of analytes can be accomplished by an electricalor mechanical pump.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the electrophoresis apparatus of thepresent invention;

FIG. 2 is an enlarged, elevated view of a plurality of analyteconcentrators stationed at the respective intersections of a large boretransport capillary and an equal plurality of small bore separationcapillaries;

FIG. 3 is an elevated view of a second embodiment of the presentinvention, showing a plurality of analytes concentrators stationed atthe respective intersections of an alternative transport channel and anequal plurality of separation channels;

FIG. 3A is an enlarged view of the described intersection containing theanalyte concentrator microstructure;

FIG. 4 is an enlarged, elevated view of an analyte concentratorstationed at the intersection of a transport capillary and a separationcapillary;

FIG. 5 is an elevated view of an analyte concentrator in the form of across-shaped capillary;

FIG. 6 is an elevated view of the electrophoresis apparatus of thepresent invention, showing an analyte concentrator disposed along thelength of a separation capillary;

FIG. 7 is a perspective view of a third embodiment of the presentinvention, showing a plurality of separation capillaries connected to asingle outlet capillary for sequential detection;

FIG. 8 is a perspective view of a fourth embodiment of the presentinvention, showing a plurality of separation capillaries adapted toanalyze multiple samples according to the techniques described in thespecification;

FIG. 9 is a perspective view of an electrophoresis apparatus having avalving system that directs the flow of fluid along a desired paththrough the transport capillary and separation capillaries;

FIG. 10 is an enlarge view of an analyte concentrator capable of beinglocalized by surrounding valves on the transport and separationcapillaries;

FIG. 11A illustrates a cross-sectional view of the analyte concentratorof FIG. 10.

FIG. 11B illustrates a cross-sectional view of the analyte concentratorwhere the transport capillary is staggered to form a analyteconcentrator that is elongated;

FIG. 12 illustrates the steps that may be taken to concentrate, isolate,and separate the desired analytes from the sample solution;

FIG. 13 is a perspective view of an electrophoresis apparatus havingvalves near the detector;

FIG. 14 is a perspective view of an electrophoresis apparatus havingtransport and separation channels with a valving system where theseparation channels merge into one output channel;

FIG. 15 is an enlarge view of one of the concentrators of FIG. 14;

FIG. 16 a perspective view of an electrophoresis apparatus havingtransport and separation channels with a valving system;

FIG. 17 is a perspective view of an electrophoresis apparatus withinlets in the separation capillaries downstream from the concentrators;

FIG. 18 is a perspective view of an electrophoresis apparatus with astaggered transport capillary forming a concentration area that iselongated;

FIG. 19 is an enlarge view of one of the concentrators with affinityelements covalently bonded to the inner wall of the separationcapillary;

FIG. 20 illustrates the process undertaken to isolate the monovalentantibody fragment Fab′;

FIG. 21 illustrates various chemical reactions used to covalentlyimmobilize an antibody or antibody fragment to the surface ofcontrolled-pore glass beads or to the surface of the inner wall of aseparation capillary, where the silanol groups of the surface of thebeads or inner wall of the separation capillary were silylated with3-aminopropyltriethoxysilane and then reacted with SSMCC before beingconjugated to a monomeric Fab′ fragment.

FIG. 22 illustrates a separation capillary having more than one type ofantibodies within its interior wall between two valves;

FIG. 23A illustrate an enlarge view of multiple antibodies along theinterior surface of a separation capillary;

FIG. 23B illustrates polymeric microstructures with Y shape antibodieshaving affinity for a particular analyte within the concentrator areawithout the need for frits;

FIG. 24A illustrates an enlarge view of multiple Fab′ fragments alongthe interior surface of a separation capillary;

FIG. 24B illustrates polymeric microstructures with Fab′ fragmentshaving affinity for a particular analyte within the concentrator areawithout the need for frits;

FIG. 25 is a perspective view of a microextraction device having fourtubing-connecting ports adapted to couple to transport and separationcapillaries;

FIG. 26 is a perspective view of the bottom side of the microextractiondevice of FIG. 25, illustrating the concentration or reaction area;

FIG. 27A illustrates the intersection area of FIG. 26;

FIG. 27B is an enlarge view of the intersection area of FIG. 27A;

FIG. 28A illustrates a microextraction device with a concentration areathat is elongated;

FIG. 28B illustrates an enlarge view of the concentration area of FIG.28A;

FIG. 29A illustrates the molecular structure of acidic drugs, ibuprofen,and naproxen;

FIG. 30 illustrates a syringe coupled to a transport tubing;

FIG. 31 illustrates a capillary electrophoresis having a concentratornear the inlet of the transport capillary;

FIG. 32A is a chart illustrating an electropherogram of ibuprofen (1)and naproxen (2), extracted by immunoaffinity bead technology from analiquot of a diluted urine specimen;

FIG. 32B is a chart illustrating an electropherogram of angiotensin II(1) and neurotensin (2) extracted by immunoaffinity bead technology froma second aliquot of the same diluted urine specimen used in FIG. 32A;

FIGS. 33A, 33B, and 33C illustrate electropherogram for analytesdetected using the electrophoresis apparatus 10 with three concentratorswith a different antibody in each of the concentrators, which actsagainst: (A) neurotensin; (B) enkephalin; and (C) cholecystokinin; and

FIG. 34 illustrates a diagnostic kit that may be used at home byindividuals to detect early signs of certain disease(s).

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates electrophoresis apparatus 10 of the presentinvention. In its elementary mode (e.g., FIG. 8), apparatus 10 performssingle sample studies on chemical or biological matrices havingconstituents or analytes of interest. But, according to the operatingprinciples shown and described, apparatus 10 can perform multipleanalyses by detecting and measuring the presence of a plurality ofanalytes (for example, three). Suitable and representative analytes mayinclude narcotics, glucose, cholesterol or pharmaceutical drugs that maybe present in urine or whole blood, as well as small and large molecularweigh substances having simple and complex structures.

As shown in FIG. 1, apparatus 10 includes platform 12 having side wall14. Sample cup 15 is mounted laterally on side wall 14. A large-bore(150-300 mm in length×500-2000 μm I.D.), nonselective introductioncapillary 16 and large-volume (1-3 ml) analyte concentrator 17 connectsample cup 15 to a first input of valve 18 which is coupled, bycapillary 20, to waste container 22 positioned on side wall 14 adjacentto sample cup 15. In a typical configuration, analyte concentrator 17comprises a matrix-like assembly of the type shown in U.S. Pat. No.5,202,010. The collective mass of the matrix is provided by largequantities of microstructures such as beads, platelets, chips, fibers,filament of the like. Individual substrates can be made from glass,plastic or other polymeric material, ceramic, or metallic compositions,and mixtures thereof. Coated or otherwise deposited onto themicrostructures are immobilized analyte-specific antibodies or otheraffinity chemistries which are suitable for characterizing andseparating particular analytes of interest. Representative antibodiesinclude those which act against peptide hormones such as insulin, humangrowth hormone and erythropoietin. These antibodies are readilyavailable from commercial vendors such as Sigma-Aldrich Co., St. Louis,Miss., and Peninsula Laboratories, Belmont, Calif.

The present invention contemplates a user-friendly, sample preparationstep which is designed to eliminate unwanted analytes that occupybinding sites and contaminate the inner walls of capillaries orchannels. This procedure will now be described with specific referenceto apparatus 10 of FIG. 2.

A first output of valve 18 is placed in the closed position and aquantity of solution from sample cup 15 is introduced into analyteconcentrator 17. Depending on its pre-selected matrix, analyteconcentrator 17 traps, in a non-specific manner, many (up to 100 ormore) different analytes, including the analytes under investigation.Sample cup 15 is then replaced by a buffer container (not shown). Thisreplacement step may be accomplished by a rotatable table mechanism ofthe type described in U.S. Pat. No. 5,045,172. Thereafter, a quantity ofbuffer is injected through analyte concentrator 17 to remove excessamounts of sample and unwanted sample components. Because valve 18remains closed during this operation, excess and unwanted samples arepassed into waste container 22.

The remainder of apparatus 10 can now be considered. A second output ofvalve 18 communicates with transport capillary 24 which, as shown byFIG. 2, intersects a plurality, here shown as three, of narrow-bore(20-75 μm) separation capillaries 28, 30 and 32. Analyte concentrators34, 36 and 38 are sequentially stationed at the intersections oftransport capillary 24 and separation capillaries 28, 30 and 32 to trapor bind different analytes of interest.

A first end (the left as viewed in FIG. 1) of separation capillary 28 isinitially placed in buffer solution cup 40. In like manner, a first endof separation capillary 30 is placed in buffer solution cup 42; and afirst end of separation capillary 32 is placed in buffer solution cup44. A major portion of separation capillaries 28, 30 and 32 extend inparallel over the upper surface of platform 12 through detection zone 45where the analytes respectively present in each of the separationcapillaries are identified by an otherwise conventional detector 46.Separation capillaries 28, 30 and 32, which terminate at groundconnection 48, may be secured to the upper surface of platform 12 byholder 49. Platform 12 can also take the form of an interchangeablecartridge with pre-positioned capillaries and analyte concentratorsproperly secured and aligned with respect to the optical system. In yetanother embodiment, best shown in FIG. 3, transport channel 24A andseparation channels 28A, 30A and 32A, having uniform and concave shapes,can be engraved, etched or otherwise formed into a glass or plasticmicrochip using known lithography or other manufacturing techniques.Analyte concentrators 34A, 36A and 38A are disposed at the respectiveintersections of transport channel 24A and separation channels 28A, 30Aand 32A as previously described.

When the sample preparation step is complete, valve 18 is opened to themain system and a buffer (e.g., sodium tetraborate) is passed throughintroduction capillary 16 and analyte concentrator 17. At this time, theanalytes of interest are released from analyte concentrator 17 using aneluting solution, along with other analyte constituents present in thesample. The analytes of interest and all the other analytes captured andreleased by concentrator 17 are passed through transport capillary 24 toanalyte concentrators 34, 36 and 38 which, as described below withreference to FIG. 3, contain a large quantity of microstructures thatare capable of binding different analytes of interest; that is, each ofthe analyte concentrators 34, 36 and 38 select and isolate a differentone of the analytes under investigation. Excess amounts of sample thenpass through the other end of transport capillary 24 to waste container27. Transport capillary 24 is subsequently washed with running bufferuntil unwanted substances are removed.

Separation capillaries 28, 30 and 32 are filled hydrodynamically(pressure or vacuum) with an appropriate electrophoresis separationbuffer which occupies the entire volume of the capillary or channel.Immobilized analytes on a solid support are stable for long period oftime. As a result, large numbers of analytes can be consequentlyseparated over time, thereby providing high throughput for the apparatusof the present invention. Separation capillary 28 is first activated byintroducing a plug of an appropriate eluting buffer from cup 40 byhydrodynamic (pressure or vacuum) or electrokinetic methods to desorb orelute analytes bound to analyte concentrator 34. The eluting buffer isimmediately followed by a freshly prepared electrophoresis separationbuffer present in replacement cup 40. Then, the power supply connectedto cup 40 is activated to begin the process of analyte separation.

As shown in Table 1, with insulin taken as representative, a typicalanalysis involves the targeted analyte of interest, its correspondingantibody, an appropriate buffer and eluting solution. TABLE 1 AntigenAntibody Separation Buffer^(\) Eluting Solution* Insulin Anti-InsulinSodium tetraborate Magnesium Chloride and Antibody (pH 8.5) EthyleneGlycol†Concentrations of electrophoresis separation buffer may range from 50mM to 200 mM.*Elution of other antigens or haptens may require a different elutingmethod. Effective eluting buffers include a 2 M solution of MagnesiumChloride and a 25% solution of Ethylene Glycol.

When the initial separation is complete, the next cycle, usingseparation capillary 30 and analyte concentrator 36, is performed in asimilar manner, i.e., the analyte is eluted from concentrator 36 andthen separated by electrophoresis migration in separation capillary 30.During these operations, the power supply is connected to one analyteconcentrator-separation capillary system at a time.

Separated analytes are then passed sequentially to detection zone 45where each analyte is recognized and measured by detector 46 using, forexample, known UV or fluorescence techniques. In one embodiment of thepresent invention, a single, bi-directional detector is indexedlaterally above platform 12 to detect analytes of interest in separationcapillaries 28, 30 and 32 or separation channels 28A, 30A and 32A. Othersub-assemblies could include a single, fixed detector and movableplatform 12 which operates to position separation capillaries 28, 30 and32 or separation channels 28A, 30A and 32A beneath the detector.Multiple detectors are movable platforms configured for X, Y and Zindexing are also contemplated.

FIG. 4 illustrates the location of analyte concentrator 34 stationed atthe intersection of transport capillary 24 and separation capillary 28.As shown in FIG. 4, and in U.S. Pat. No. 5,202,010, porous end plates orfrits 50, which permit fluid flow, are provided in transport capillary24 and separation capillary 28 to act as barriers for holdingmicrostructures 54 in analyte concentrator 34.

Alternatively, as shown in FIG. 5, analyte concentrator 55 can befabricated by using two constricted areas with no frits at all. Analyteconcentrator 55, in the form of a cross-shaped capillary, has innerdiameter 61 and 63 preformed in relation to inner diameter 57 oftransport capillary 24 and inner diameter 59 of separation capillary 28.

Analyte concentrator capillary 55 contains a plurality of previouslydescribed microstructures 54 which are larger than inner diameters 57and 59. They are typically coated with non-specific chemistries such asC-18 or highly specific antibodies or antigens having an affinity forone of the analytes under investigation. Several other well-knownchemistries can also be used.

In the embodiment illustrated by FIG. 5, microstructures 54 are retainedor confined in the interior of analyte concentrator 55 by making innerdiameter 57 of transport capillary 24 smaller than inner diameter 61 ofanalyte concentrator 55. In like manner, inner diameter 59 of separationcapillary 28 is smaller than inner diameter 63 of analyte concentrator55. For example, inner diameters 57 and 59 may be one-quarter toone-half the size of inner diameters 61 and 63.

To increase detection sensitivity for a particular analyte, achromophore may be added to the eluting buffer to elute and tag thebound analyte for the purpose of enhancing the ultraviolet absorptivity,fluorescence, phosphorescence, chemiluminescence or bioluminescence ofthe analyte as it passes through detector 46.

In an alternative technique to increase detection sensitivity,additional analyte concentrator 60 may be placed in one of separationcapillaries 28, 30 and 32, as shown in FIG. 6. Analyte concentrator 60has a plurality of microstructures 54 coated with a chromophoric agentor antibody that binds to a portion of a chromophoric agent whichincreases ultraviolet absorptivity, fluorescence or phosphorescence whenbound to a minute quantity of a specific analyte. Frits 62 are locatedat the input and output of analyte concentrator 60, and narrow capillary64, which interests with separation capillary 28, carries a buffer toperiodically cleanse microstructure 54 in analyte concentrator 60 aftereach analysis.

An analyte tagged with a chromophoric agent is more readily identify bythe apparatus of the present invention, thereby increasing thesensitivity of analyte detection by as much as 100 times or more. Manydifferent chromophoric agents emit light when they bind a specificfunctional group to form a product molecule in an electronically excitedstate.

The alternative embodiment illustrated by FIG. 7 is similar to thatshown in FIG. 1. But, the FIG. 7 embodiment is different because theoutput ends of separation capillaries 28, 30 and 32 are connected toeach other at the interface with a single outlet capillary 66 whichcooperates with on-column detector 86 that senses ultraviolet (UV) orfluorescent energy. The exit position of outlet capillary 66 may also beconnected (as shown) to off-column detector 88 which comprises anelectrochemical, mass spectrometry, circular dichroism detector ornuclear magnetic resonance detector.

The electrophoresis apparatus of FIG. 7 employs multiple separationcapillaries or channels for sample concentration, but only one outletcapillary for sample detection. This coordinated separation byindividual capillaries may be sequentially activated and controlled bywell-known electronic circuitry. Like the FIG. 1 embodiment, precedinganalytes are completely separated and detected before the nextseparation operation is activated.

The electrophoresis apparatus of FIG. 8 is similar to that of FIG. 7,but it is adapted to work with multiple samples (here, e.g., three)having a simple or complex component. There is no introduction(transport) capillary 16 or sample cup 15 as provided by embodiments ofFIG. 1 and FIG. 7. Separation capillaries 28, 30 and 32 are equippedwith single analyte concentrators 34, 36 and 38, respectively.Individual samples are directly and sequentially delivered to separationcapillaries 28, 30, 32, and the analytes of interest are captured usingsuitable chemistries as previously described. The capillaries may bewashed with buffer until all salts and unwanted substances are removed.Like the FIG. 7 embodiment, separation capillaries 28, 30 and 32 areactivated in series one after the other. When all the analytes areseparated in a single capillary, the apparatus begins the nextseparation cycle in the next capillary. In each of the describedembodiments, apparatus 10 provides greater efficiency and higherthroughput when compared to prior art devices.

Improved instrumentation containing a series of solid-phasemicroextraction devices on-line in a multi-dimensional electrophoresisapparatus has been developed for selective and non-selective molecularconsolidation and it is described in U.S. Pat. No. 6,406,604 B1, whichis hereby incorporated by reference. These devices, known as analyteconcentrators or analyte concentrators-microreactors containing affinityprobes to bind target compounds, permit the capturing of analytespresent in simple or complex mixtures for purification, desalting andenrichment purposes. Furthermore, it allows the performance of manychemical and/or biochemical reactions, such as the on-line enzymaticcleavage of proteins to generate peptides. This continuation-in-partdescribes further improvements of the described embodiment.

FIG. 9 illustrates an electrophoresis apparatus 10 including a valvingsystem 100 that directs the flow of fluid along a desired path throughthe transport capillary 24 and separation capillaries 28, 30, and 32. Inthis example, valves 102, 104, 106, and 108 may control the flow ofbuffer solution(s) around the concentrator 34; valves 106, 110, 112, and114 may control the flow of buffer solution(s) around the concentrator36; and valves 112, 116, 118, and 120 may control the flow of buffersolution(s) around the concentrator 38. With the valving system 100, theenvironment for each of the concentrator may be localized. Localizing aconcentrator allows for independently controlling the microenvironmentof that concentrator, such as controlling the concentration of reagents,temperature, time of reactions, etc. The valving system 100 allows theloading of one or more appropriate background electrolyte solutions, theintroduction of the samples to be analyzed by the various modes ofcapillary electrophoresis, and the cleaning of the capillaries so thatthe capillaries may be reused.

The transport capillary 24 and the separation capillaries 28, 30, and32, along with the valving system 100 may be incorporated into theplatform 12 of the electrophoresis apparatus 10 in a variety of ways.For instance, holders 49 may be used to hold the capillaries in placerelative to the platform 12. After certain number of usage, thecondition of the capillaries or the valving system 100 may degrade sothat they may need to be replaced. In such instances, the holders 49 maybe removed from the platform 12 and a new system of capillaries andvalving system may be installed into the platform. Alternatively, a newsystem of capillaries and valves may replace the existing capillaries toisolate different types of analytes from the sample solution in the cup15. The concentrators 34, 36, and 38 in the replacement capillaries mayeach have different immobilized affinity ligands that are attracted to adifferent type of analyte than the ones they are replacing. This way,the electrophoresis apparatus 10 may be reused and adapted to isolate avariety of analytes.

The transport capillary 24 may be also adapted to perform isoelectricfocusing (IEF) separation of a sample solution by maintaining the valveson the transport capillary opened and the valves on the separationcapillaries closed. The intersection of the transport and separationcapillaries may be emptied without frits and matrix-assembly in theconcentrators. IEF is a method of determining the isoelectric point (pI)of a protein by carrying out electrophoresis in a capillary or gelcontaining a pH gradient. The pI is the pH at which a protein will notmigrate in an electric field and is determined by the charged groups inthe protein. Proteins can carry positive, negative or zero chargedepending on their local pH, and for every protein there is a specificpH at which its net charge is zero; this is its pI. IEF utilizesdifferent pI in proteins to separate the proteins based on their pIlevels. When a protein is placed in a medium with a pH gradient andsubjected to an electric field it will initially move towards theelectrode with the opposite charge.

During migration through the pH gradient the protein will pick up orlose protons. As it migrates the net charge and the mobility willdecrease and the protein will slow down. Eventually the protein willarrive at the point in the pH gradient which is equal to its pI. At suchpoint, the protein will be uncharged and stop its migration. If theprotein should happen to diffuse to a region outside its pI it will pickup a charge and hence move back to the position where it is neutral. Inthis way proteins are condensed, focused, or separated into certainbands according to their pI levels. This way, dual mode of separationsmay occur with the electrophoresis apparatus 10, IEF separation throughthe transport capillary and the separation of the desired analytethrough the separation capillaries. In this case, one electrode may beprovide in the cup 15 and the other electrode on the outlet end of thetransport capillary to provide the electric field to focus and separatethe proteins present in transport capillary 24. After isoelectricfocusing separation is completed, the valves on the transport capillarymay be closed and the valves on the separation capillaries may beopened. Further separation of the proteins may be accomplished by othermodes of capillary electrophoresis in separation capillaries 28, 30, and32.

FIG. 10 illustrates a perspective view of the valving system 100 for oneof the analyte concentrators. Each concentrator may be surrounded byfrits or porous end plates 35 provided along the path of the transportcapillary 24 and the respective separation capillary to retain thematrix-like assembly 37 within the concentrator. The valves on thetransport capillary and the separation capillary also surround each ofthe concentrators to control the flow of sample solution through thetransport capillary 24 and through the respective separation capillary.The valves may be motor operated, that is controlled remotely by aprocessor based on a predetermined set of instructions such as asoftware program. After the concentrators 34, 36, and 38 have beenproperly conditioned, the valves along the transport capillary may beopened and the valves along the separation capillaries 28, 30, and 32may be closed to allow the concentrated sample solution from theconcentrator 17 to pass through the concentrators 34, 36, and 38. Thisallows each of the matrix-like assembly in the concentrators 34, 36, and38 to bind to the desired analyte from the concentrated sample solution.The remaining concentrated sample solution may be released to the wastecontainer 27 on the other end of the transport capillary 24.

Once each of the desired analytes of interest are bound to therespective matrix-like assembly within the concentrator, the valves onthe transport capillary may be closed and the valves on the separationcapillaries 28, 30, and 32 may be opened. To separate the desiredanalyte(s) that are attached to each of the matrix-like assembly in theconcentrators 34, 36, and 38, a separation solution may be passedthrough the separation capillaries 28, 30, and 32 so that each of thedesired analyte may travel towards the detection area 45 after releasedfrom the concentrators 34, 36, and 38. More detail steps involved in theprocess of concentrating, isolating, and separating the desired analytesfrom the sample solution provided in the sample cup 15 are discussedlater in the specification.

FIG. 10 illustrates a concentrator with porous end plates or frits 35,which permit fluid flow, in the transport capillary 24 and separationcapillary 28 to act as semi-permeable barriers for holding matrix-likeassembly 37 within the analyte concentrator. For the concentrator 34,the frits 35 may be formed along the transport capillary 24 and theseparation capillary 28. The frit 35 and the matrix-like assembly 37 maybe the type shown in U.S. Pat. Nos. 5,202,010 and 6,406,604, which arehereby incorporated by reference. The matrix-like assembly may beprovided in many forms. For instance, the collective mass of the matrixmay be provided by large quantities of microstructures such as beads,platelets, chips, fibers, filaments, monolithic polymers, sol-gel, orthe like. Individual substrates can be made from glass, plastic,ceramic, metallic, or any appropriate polymeric chemistry compositions,and mixtures thereof. The use of interconnected beaded and/or polymericmicrostructures may not require the presence of frit structures to holdthe matrix, because they form a net that is linked by chemical bonding,and they are usually positioned in a rigid configuration. In most cases,these beaded or polymerized microstructures may sustain low-pressures.However, in certain cases where high pressures may be needed, theirnetwork configuration can be deteriorated or destroyed. Covalently ornon-covalently affinity ligands coated or immobilized onto the surfaceof the beaded microstructures or monolithic polymers, sol-gel, ordirectly onto the inner wall of the capillary, are immobilizedanalyte-specific antibodies or other affinity chemistries which aresuitable for characterizing and separating particular analytes ofinterest. Representative antibodies include those which act againstpeptide hormones such as insulin, human growth hormone and a variety ofantibodies directed against any substance of small molecular weight(classified as hapten) or any substance of larger molecular weight orbiopolymer (classified as antigen). These antibodies are readilyavailable from commercial vendors such as Sigma-Aldrich Co., St. Louis,Mo., and Peninsula Laboratories, Belmont, Calif., and many othercompanies worldwide. Alternatively, one skilled in the art maymanufacture a desire monoclonal and/or polyclonal antibody byconventional methods or protocols described in the literature. Not allhaptens are capable of elicit an antigenic response by itself, usuallythey need to be bound to an antigenic protein carrier to generate anantibody.

The matrix-like assembly may include affinity elements immobilized invarious configurations and orientations in order to obtain a higherconcentration of the desired analytes. For example, antibody fragmentsmay be used instead of complete antibodies to obtain a higherconcentration of the desire analytes. The larger diameter of thetransport capillary 24 may require that the two frits in capillary 24 belarger than the frits in the separation capillaries 28, 30, and 32.Conversely, the matrix-like assembly may be configured to capture thedesired analytes through the use of affinity ligands that areimmobilized onto the surface of frit-free polymeric structures, asmentioned above. Alternatively, affinity ligands may be immobilized ontothe surface of commercially available magnetic beads to be used asmatrix material and substantially confined to a predetermined locationwithin the capillary through magnetic attraction. Using magneticattraction to hold the matrix in a predetermined location along thecapillary may eliminate the need for frits. The absence of the frits mayallow the flow of sample through the capillary to move faster, whileretaining a surface to attach the affinity elements.

The concentrator 17 may include immobilized ligands comprised of asingle nonselective or a mixed-mode non-selective type of chemistriessuch as reversed-phase C18 and ion-exchanger matrices or resins, etc.The mixed mode may be allowed to capture and enrich a wide range ofanalytes based primarily on their physico-chemical properties, includingthe charge, size, shape, hydrophobicity, etc. The reversed-phase C18chromatography adsorption resins, anion exchange matrices or resins,cation exchange, immobilized metal affinity capture, or mixed-modesresins may be placed in the concentrator 17 in a sequential order, onetype first and then the other, or as a mixed matrix. The analytes canalso be eluted in a sequential order according to their physico-chemicalproperties.

The concentrator 17 may also be composed of immobilized ligandsincluding a selective type of chemistry such as antibody, lectin,enzyme, aptamer, dye affinity chromatography, etc. For example, aparticular lectin can recognize a specific sugar in a sugar-containingelement, such as a glycoprotein, and retain the entire glycoproteinmolecule. The selective type of chemistry may bind a single analyte or avery close structurally related analyte. In the case of a completemonomeric antibody, it may have two antigen-binding sites; in the caseof a Fab fragment, it may have a single antigen-binding site. However,in the case of other selective affinity ligands, it may have more thanone site to bind the target analyte, an enzyme may have an active siteto bind the corresponding substrate, and an inhibitor-activator may bindto the same active site or to a different site (e.g., allosteric site).The concentrators 34, 36 and 38 may also include immobilized affinityligands other than antibody fragments, as described above forconcentrator 17. Proteolytic enzymes may be immobilized to theanalyte-concentrator-microreactor to carry out microreactions, such asthe cleavage of a protein into peptide components. In the microreactoror bioreactor, a number of chemical and/or biochemical reactions can beperformed involving a large number of affinity ligands to be immobilizedto the microreactor. For example, peptide synthesis, nucleic acidsynthesis, small molecular weight substances synthesis can beaccomplished in a small scale. The entrapment of viruses, cells, orsubcellular structures may also be used to study metabolic pathways anddegradation products of small molecular weight substances, as well asbiomolecules.

The concentrator 17 generally includes matrix-like assembly or resinmaterial that captures a larger number of analytes as well as a greatervariety of analytes than the concentrators 34, 36, and 38. Theconcentrators 34, 36, and 38 may include corresponding matrix materialincluding high-specificity immobilized affinity ligands that may be moreselective than the matrix material including non-specific immobilizedaffinity ligands used in the concentrator 17. As a consequence, thematrix in the concentrators 34, 36, and 38 may capture or isolate asmaller quantity of analytes than the concentrator 17, but moreselective and pure desired analytes, so that the captured analytes aremore concentrated than in the original biological fluid cell, tissue,organ, or any other simple or complex matrix. The selectivity of theconcentrator 34, 36 and 38 comes from the antibody capable ofrecognizing a specific area in a particular analyte called the epitope(e.g., a monoclonal antibody recognize a single epitope, a polyclonalantibody recognized multiple epitopes). Some analytes may have abundantamount of sugars or additional components on the surface of the molecule(e.g., certain glycoproteins) that may hinder the binding process to aspecific peptide sequence. To better enable the capture of complexanalytes, such as bulky and complex biomolecules, concentrator 34, 36,and 38 may contain two or more affinity ligands components. For example,a combination of a specific antibody and a specific lectin may be placedinside the concentrator to be able to capture a particular type ofanalyte through a selective peptide and/or epitope or through aselective sugar present on the analyte or to both. The specificattraction of each component to a different portion of the analyte mayincrease the number of complex analytes being attached.

FIG. 11A illustrates a cross-sectional view of FIG. 10 where the valveson the transport capillary are in the second or closed position tosubstantially prevent the sample solution from passing towards theconcentrator. The valves on the separation capillary are in the first oropen position to allow the buffer solution to pass through theconcentrator. The frits 35 surrounding the concentrator substantiallyretain the matrix-like assembly 37 within the concentrator.

FIG. 11B illustrates that the transport capillary 24 may be staggeredfrom one separation capillary to another to form a concentration area 34that is elongated. This allows additional matrix-like assembly 37 to beincorporated into the concentration area 34 to attach a desired analytefrom the sample solution. In addition, the sample solution may take moretime to pass through the elongated concentration area 34, which allowsthe matrix-like assembly additional time to bind to the desired analytefrom the sample solution. The concentration area 34 may be surrounded byfrits or porous end plates 35 to retain the matrix-like assembly 37within the concentration area 34.

FIG. 12 illustrates the steps that may be taken to concentrate, isolate,and separate the desired analytes from the sample solution provided inthe sample cup 15. A first conditioning step 101 prepares the transportand separation capillaries to a desired condition. This may beaccomplished by passing conditioning buffer solution through thetransport and separation capillaries. The conditioning step 101 mayimprove the binding properties for the immobilized affinity ligands sothat once the desired analyte is attracted, it is retained by theimmobilized affinity ligands for as long as the optimized conditions aremaintained. The conditioning buffer solution may be provided through thetransport capillary 24 and/or the separation capillaries 28, 30, and 32.

Once the capillaries have been conditioned with a conditioning buffer orsolution, the sample solution in the cup 15 may be introduced throughthe transport capillary 24. For a large capacity concentration step 103,the valve 18 may be closed and the concentrator 17 used to obtain theconcentrated sample of desired analytes. The concentrator 17 may havemore surface area for greater capacity to capture the desired analytesthan the other concentrators used in the valving system 100. In general,the concentrator 17 may be used for more complex matrices where severalanalytes may be present in the sample. For instance, the concentrator 17may be used when hundreds or thousands of analytes are present in thesample. On the other hand, when isolating certain compounds present insimple matrices, there may not be a need for the concentrator 17, tube20, and waste cup 22 (depicted in FIG. 9). Examples of simple matricesinclude microdialysates, artificial matrices containing standardcompounds, etc. In such instances, the samples solution may beintroduced directly to transport capillary 24 from the cup 15 containingthe simple matrix.

The isolation or concentration of the desired analytes may be done in adifferent location and time. The concentrated analytes may then beprovided to the transport capillary 24 at a later time. The independenceof the concentrator 17 from the apparatus 10 allows the concentrator 17to be removed and replaced with a new concentrator without altering theapparatus 10. In addition, a plurality of original samples may beprovided in a plurality of cups that are positioned along a rotatabletable or through an appropriate fraction collector or the like, toprovide the sample solution in each cup to the transport capillary 24 inintervals as the table rotates or moves, thereby providing multiplesamples to the transport channel 24. Similar rotatable table may be usedto change buffer solutions present in cups 40, 42, and 44.

After the sample solution has been introduced into the transportcapillary 24 and passed through concentrator 17, in step 105, theconcentrator 17 may be cleaned. This may be accomplished by passingcopious amounts of cleaning buffer to the concentrator 17 followed byconditioning buffer from another cup 15′, replacing cup 15, throughcapillary 20 and towards waste cup 22. At this stage the compounds boundto concentrator 17 can be removed or eluted out of the concentrator 17.In the elution step 107 of FIG. 12, analytes retained by theconcentrator 17 can be eluted from the concentrator 17 in many ways. Oneway is to pass a small amount or plug of an appropriate elution ordesorption solution through the concentrator 17 to remove the boundanalytes to the transport capillary 24. The bound analytes from theconcentrator 17 are passed through the transport capillary 24 so thatthe concentrators 34, 36, and 38 may further isolate the desiredanalytes in each of the concentrators 34, 36, and 38. The removal of thebound compounds can be carried out as a group (simultaneously), or oneor more at the time (stepwise or sequential). For isolating the desiredanalytes, which are cleaner or more pure and more concentrated than theoriginal sample solution, provided in the sample cup 15, a plurality ofconcentrators containing more selective affinity ligands in this matrixmay be used, such as concentrators 34, 36, and 38 along the transportcapillary 24 with the purpose of individually capturing a single or amore reduced number of compounds than those bound to the concentrator17. Accordingly, there may be two concentration steps in the invention:in the first concentration step, the concentrator 17 may be used toclean or purify the sample solution from a complex mixture; and in thesecond concentration step, the cleaned sample solution is passed throughthe concentrators 34, 36, and 38 to isolate the desired analyte(s) intoeach of the concentrators 34, 36, and 38 to isolate the desiredanalyte(s) that is different than the other.

To allow the sample solution to flow through the concentrators 34, 36,and 38, the valves 18, 102, 106, 112 and 118 along the transportcapillary 24 may be opened; but to prevent the sample solution fromflowing through the separation capillary, the valves 104, 108, 110, 114,116, and 120 along the separation capillaries may be closed so that thesample solution does not flow to the buffer solution cups 40, 42, and44, nor towards the detection system. Each of the concentrators 34, 36,and 38, may be filled with matrix-like assembly that are free-floatingor chemically bonded microstructures, or polymeric monolithic matrices,containing appropriate selective and/or non-selective affinitychemistries. The concentrators may contain frit structures or befritless.

As the sample solution passes through the concentrators, each of theconcentrators may isolate the desired analyte(s) from the samplesolution as discussed above. The excess sample solution may pass throughthe other end of the transport capillary 24 to the waste container 27.To optimize the binding, the valves 102 and 118 may be closed alongtransport capillary 24, to allow the analytes present in the samplesolution to have a longer period of time to be exposed to thematrix-like assembly with corresponding immobilized affinity ligandsbound to the particles or microstructures in each of the concentrators34, 36 and 38. Alternatively, an elongated concentration area 34 asdisclosed in FIG. 11B may be provided to expose the sample solution tothe matrix-like assembly for a longer period of time and a longersurface area to capture larger amounts of desired analyte(s).

With the valving system 100, each of the concentrator areas may belocalized so that an appropriate temperature, for example, may becontrolled to each of the concentrator areas to improve the conditionfor the desired analyte to bind to the immobilized affinity ligands inthe respective concentrators 34, 36, and 38. The desired temperature forthe binding to occur may vary for each analyte. For example, the desiredtemperature may be at 25 C rather than at 37° C., or vice-versa, or evenhigher or lower than these temperatures. Each concentrator may have anindependent temperature control to optimize the binding. In someinstances, a gently shaking or use of a microwave pulse or acousticmicromixing system may aid in the binding process. For example, the useof a microwave pulse can accelerate the work of proteases and reduce thetime required to cleave a protein into its peptide components.

With the desired analytes isolated in the concentrators 34, 36, and 38in step 107, the isolated analytes in the concentrators 34, 36, and 38may be cleaned, in the cleaning step 109. The cleaning step 109 removesremaining salts and unwanted materials present in the enriched samplesolution passed from concentrator 17. This may be done by passing thecleaning solution through transport capillary 24 or through theseparation capillaries. The cleaning solution washes away at least someof the salts and unwanted materials while the immobilized affinityligands in each of the concentrators 34, 36, and 38 maintain its bind onthe desired analyte(s). The cleaning step 109, however, may weaken thebinding properties for the immobilized affinity ligands in theconcentrators 34, 36, and 38. As such, once concentrators are clean, asecond conditioning step 111 of the capillaries may be provided to onceagain improve the binding properties of the immobilized affinity ligandsin the concentrators 34, 36, and 38. The separation capillaries 28, 30and 32 may be conditioned until they are equilibrated with aconditioning buffer present in cups 40, 42 and 44.

In the second elution step 113, the elution buffer is used for releasingthe desired analyte from the immobilized affinity ligands in theconcentrators 34, 36, and 38. The amount of a plug of elution bufferthat is needed to release the desired analyte from the immobilizedaffinity ligands may vary. In general, about 50 to about 200 nanolitersof the elution buffer may be used. Also, as the size of the internaldiameter of the capillary increases, greater amount of the elutionbuffer solution may be used. The condition of elution buffer may begentle as possible so that the capturing properties of the immobilizedaffinity ligands remain intact in the surface of the particles ormicrostructures, or in a portion of the inner wall of the capillary sothat it may be reused.

In the separation step 115, the separation buffer is used to separatethe analyte(s) released from the concentrators. The separation buffermay be provided through cups 40′, 42′ and 44′. In some instances, theconditioning buffer and separation buffer may be the same. Thecomposition of each conditioning and separation buffer for eachseparation capillary may be the same or different. For the conditioningand separation step, the valves 102, 106, 112, and 118 on the transportcapillary 24 may be closed and valves 104, 108, 110, 114, 116, and 120on the separation capillaries 28, 30 and 32 may be open. At this stagethe desired analytes bound to the concentrators 34, 36, and 38 may bereleased sequentially or simultaneously using a small plug of desorptionsolution. If analytes are released in a sequential order, they can bereleased from concentrators 34, 36, and 38 in any order. For example, torelease the analyte(s) retained by the concentrator 36 first, the valves110 and 114 are opened first with the valves 106 and 112 being closed.As mentioned above, this allows three buffer systems to be introduced tothe separation capillary 30 from cup 42, creating an independentoptimized microenvironment of conditioning, desorption and separation.The first buffer is a conditioning buffer. The second buffer is aseparation buffer. The third buffer is a small plug of an elution ordesorption buffer. The separation capillary can be temperaturecontrolled where the separation capillary has a linear, coiled,serpentine configuration. In addition, each separation capillary mayhave a different configuration.

The buffers in the cup 42 may be changed using a variety of methods. Forexample, an autosampler, rotatable table or any other manual orautomated device that holds a plurality of sample containers, vials, orcups, may be used. For instance, three cups may be needed for holdingthree different buffers, vials 42 (conditioning buffer), 42′ (separationbuffer), and 42″ (elution buffer). For the separation step, aplatinum-iridium electrode can be introduced to the cup 42 (high voltageside) containing the separation buffer. The electrode may, in turn, beconnected to a high-voltage cable and a high-voltage power supply. Onthe opposite side of the separation capillary 30, a grounding electrodemay be provided for grounding. When the power supply is switched on, thesystem is activated to begin the process of releasing and separating theanalyte(s). The process of desorption or elution of the analyte(s) bythe chemical constituents of the small plug of the elution buffer canoccurs by moving the plug by pressure, or vacuum, or electrokinetically.Similar steps may be taken to release the analytes in the concentrators34 and 38 in any order. For instance, to release the analyte isolated inthe concentrator 34, the valves 102 and 106 may be closed and the valves104 and 108 opened. Similar to a concentrator, each individualseparation capillary 28, 30, or 32 may have an independently controlledtemperature system. The capillary can be heated or cooled in a linearformat or in a coiled configuration using a controlled-temperature fluidor device such as a Peltier.

As the analytes in the concentrators 34, 36, and 38 are released in apredetermined order, the detector 46 of FIG. 1 may be movable andaligned with the separation capillary corresponding to the concentratorthat the analyte is released from. For instance, with the above example,if the analyte from the concentrator 36 is released first, then thedetector 46 is first aligned with the separation capillary 30 toidentify the analytes released from concentrator 36. Then, the detector46 may be repositioned to align with the separation capillary 28 todetect the analytes released from the concentrator 34, and repositionedto detect the analytes passing through capillary 32 released fromconcentrator 38.

The valving system may communicate with a detection system for detectingthe analytes released from the concentrators. The detecting system mayoperate in many ways. For instance, the detection system may include adetector for each separation capillary 28, 30, and 32. In anotherembodiment, the three separation capillaries may be merged into one exitcapillary as shown in FIGS. 7 and 8, and one detector is aligned overthe exit capillary. In this case, the detection system may have onedetector that is fixed such that it can align over the detection windowpositioned in the exit capillary 66 for detecting the analytes passingthrough the exit capillary. For this operation, however, additionalvalves may be needed to direct the separated analytes from separationcapillaries 28, 30, and 32 to the single detector. For example, whenseparation capillary 28 is active and analytes are separated withincapillary 28, capillaries 30 and 32 may be inactivated, and theseparation buffers may be blocked by the corresponding valves. The fixeddetectors, 86 and 88, of FIGS. 7 and 8 may be a laser-inducedfluorescence detector or a contactless electrochemical detector or acombination of similar detection devices. Furthermore, the outlet of theexit capillary may be connected to other detector systems, such as amass spectrometer, including sample deposition onto a matrix assistedlaser desorption/ionization (MALDI) plate, or a conductivity detector.

The analytes in the concentrators 34, 36, and 38 may be releasedsimultaneously as well. This may be accomplished by closing the valves102, 106, 112, and 118 along the transport capillary 24 and opening thevalves 104, 108, 110, 114, 116, and 120 along the separation capillaries28, 30, and 32. As the analytes in the concentrators 34, 36, and 38 arereleased simultaneously through the separation capillaries 28, 30, and32, the detection of the separated analytes may be accomplished asdescribed above. The capillary electrophoresis separation of theanalytes in capillaries 28, 30, and 32 may require a single power supplywith the appropriate high-voltage relays or multiple power supplies,each for a single column. With the valving system 100, the path thatsample and buffer solutions flow through the transport capillary 24 andthe separation capillaries 28, 30, and 32 may be controlled to localizethe concentrators so that a customized environment for each analytebound to the microstructures in the analyte concentrator may be formed.The separation of the analytes can occur using electricity(electroosmotic flow), controlled positive pressure or vacuum, or acombination of both.

FIG. 13 illustrates an electrophoresis apparatus 10 including a valvingsystem 100 having valves 121, 123, and 125 on the separation capillaries28, 30, and 32, respectively, near the detection window 45. The outputends of the separation capillaries 28, 30 and 32 may be connected toeach other at the interface with a single outlet capillary 66 whichcooperates with on-column detector 86 that senses ultraviolet (UV) orfluorescent energy. The outlet of the outlet capillary 66 may also beconnected (as shown) to a waste container 48. With the valves 121, 123,and 125, the analytes in the separation capillaries may be released tothe output capillary 66 sequentially by opening one valve at a time.This allows the analytes in the concentrators 34, 36, and 38 to bereleased simultaneously but sequentially detect the analytes in each ofthe concentrators through the valves 121, 123, and 125. In addition, thevalves 121, 123, and 125 may be synchronized with the valves surroundingthe concentrators 34, 36, and 38 to release the analytes in theconcentrators 34, 36, and 38 in a predetermined order.

FIG. 14 illustrates that the transport channel 24A and separationchannels 28A, 30A and 32A, for the electrophoresis apparatus 10 may beformed with uniform and concave shapes that are engraved, etched orotherwise formed into a glass or plastic microchip using knownlithography or other manufacturing techniques. Analyte concentrators34A, 36A and 38A are disposed at the respective intersections oftransport channel 24A and separation channels 28A, 30A and 32A with thevalving system 100 to control the flow of fluid and microenvironment toeach of the concentrators 24A, 36, and 38 as previously described. Nearthe detector 66, valves may be provided to control of fluid to theoutput capillary 66 from the plurality of separation capillaries. FIG.15 illustrates that each concentrator formed by the intersection oftransport and separation channels may be surrounded by valves to controlthe flow of liquid through the transport channel 24A and thecorresponding separation channel.

FIG. 16 illustrates a perspective view of an electrophoresis apparatus10 having a transport channel 24A and a plurality of separation channels28A, 30A, 32A, and etc. Near the outlet side of the separation channels,a detector 86 may be provided that aligns with one of the detectionwindows of the separation channels to detect the analyte passing throughthe respective separation channels sequentially. To simultaneouslydetect the analytes passing through all of the separation channels, adetector may be provided for each separation channel to speed up theprocess.

FIG. 17 illustrates that the new separation buffer solution may be addedby auxiliary capillaries 122, 124, and 126 after or downstream from theconcentrators in order to preserve the integrity of the antibody or anyother immobilized affinity ligands. In certain applications the analytesunder study may require for optimal separation from a separation buffersolution that may adversely affect the activity of the intact antibody,antibody fragment, lectin, enzyme, or any affinity ligands affected bycertain compounds present in the separation buffer. Put differently,with certain separation buffer solutions may adversely affect thebinding property of the immobilized affinity ligands in theconcentrators so that the affinity ligands may not be used again. Also,the analytes may not be retained by the immobilized affinity ligands.With the auxiliary capillaries 122, 124, and 126, the separation buffersolution may be introduced into the separation capillaries using thecups 128, 130, and 132. This allows the separation buffer solution toflow towards the detecting zone so that there is minimal, if any,interaction between the separation buffer solution and the antibody inthe concentrator. For example, the separation of an analyte may requirethe presence of organic solvents or other additives in the separationbuffer solution such as urea, certain detergents, etc. If suchseparation buffer solution passes through the concentrator so that theseparation solution interacts with the antibody in the concentrator, theseparation buffer solution may disrupt the binding process between theanalyte and the antibody during the conditioning process of thecapillary and/or destroy the quality of the antibody in an irreversiblemanner. Such adverse effect on the antibody may destroy the integrity ofthe binding capacity of the antibody so that it may not bind to theanalyte and/or may not be used again. To substantially prevent suchadverse effect on the antibody, the antibody in the concentrator isisolated from such separation buffer solution to protect the immobilizedantibody, or antibody fragments or other affinity element, such as alectin or an enzyme.

In addition, the binding and separation conditions of a desired analytemay require different optimization conditions. In cases where theconditioning and/or separation buffer are different, one or more of theseparation capillaries 28, 30, and 32 may be divided into two stages. Inthe first stage of the conditioning process, capillaries 28, 30, and 32may be filled with the appropriate conditioning buffer located in thecups 40, 42, and 44, respectively, to improve the binding condition forthe antibody. The conditioning buffers in the respective cups may passthrough the open valves 104, 110, and 116, and pass through theconcentrators 34, 36, and 38, and pass through the valves 108, 114, and120, and then to the outlets of the separating capillaries. The valves102, 106, 112, and 118 along the transport capillary may be closed tokeep the conditioning buffer within each of the separation capillaries.

FIG. 17 illustrates cups 128, 130, and 132 located on the second stageof the separation capillaries 28, 30, and 32. The cups 128, 130, and 132may be coupled to the corresponding separation capillaries throughauxiliary capillaries 122, 124, and 126, respectively. The cups 128,130, and 132 may hold separation buffer solutions that are fed into theseparation capillaries 28, 30, and 32 downstream from the concentrators34, 36, and 38, respectively. The auxiliary capillaries 122, 124, and126 used to couple the cups 128, 130, and 132 to the separationcapillaries 28, 30, and 32 may be electrolyte-provider capillaries(EPCs). The auxiliary capillaries 122, 124, and 126 may be coupled tothe respective separation capillaries 28, 30, 32, downstream or afterthe concentrators 34, 36, and 38 so that the buffer solutions flowtowards the detecting window 45. The auxiliary capillaries 122, 124, and126 may be also coupled to the valves 108, 114, and 120 downstream fromthe concentrators 34, 36, and 38 to control the flow of the buffersolution into the separation capillaries 28, 30, and 32 by opening andclosing the valves 108, 114, and 120. This way, the buffer solutionsgenerally do not interact with the immobilized antibodies in theconcentrators 34, 36, and 38. With the cups 128, 130, and 132 positioneddownstream from the concentrators in the apparatus 10, the separationbuffer may be introduced into the apparatus 10 either before theconcentrators using the cups 40, 42, and 44, or after the concentratorsusing the cups 128, 130, and 132, depending on the interfering of theseparation buffer on the binding between the analyte(s) of interest andthe immobilized affinity ligands in the concentrators 34, 36, and 38and/or the damage that the constituents of the separation buffer can doto the immobilized affinity ligands.

In applications where the separation buffer does not adversely affectthe antibody, the separating buffer solution may be introduced into theseparation capillary before the concentrator through the cups 40, 42,and 44 as discussed above. For applications where EPCs are used, theconcentration step is similar to the step discussed above. For theeluting and separating steps, the valves on the separation capillaries28, 30, and 32 may be opened sequentially or simultaneously to performthe process of simultaneous elution and separation of the analytespresent in all of the concentrators and separation capillaries, thevalves along the transparent capillary 24 may be closed, and the valves104, 110, and 116 along the separation capillaries 28, 30, and 32 may beopened first. The eluting buffer solution flows through the separationcapillaries 28, 30, and 32 to elute the analytes bound to the antibodiesin the concentrators 34, 36, and 38, respectively. This causes theanalytes to be released from the immobilized antibodies or antibodyfragments, or other affinity ligands.

For the separating step in which a separation buffer for optimizedseparation of the analytes is needed, but may cause disruption of thebinding between the analyte and affinity ligands or may damage theintegrity of the affinity ligands, the valves 108, 114, and 120 may beopened to allow the separation buffer solutions in the cups 128, 130,and 132 to allow an optimized separation of the release analytes downstream from the concentrators. The separating buffer solution may enablethe separation of the analytes under improved conditions so that oneanalyte or other closely related analyte(s) that have selectively boundto the immobilized ligands may be separated achieving a based-lineresolution after elution from their respective analyte concentrators.

FIG. 18 illustrates another embodiment of electrophoresis apparatus 10,configured to capture and detect primarily large sized particles such ascells, organelles, and/or other bulky globule structures. The largeparticles may require a larger cross-sectional area for the particles topass through without blockage or interference during separation. Theconfiguration where the affinity ligands are immobilized on the surfaceof a bead, or cross-linked, or on monolithic structures may not beappropriate for the separation of globule structures. The blockage mayoccur in such situations and may prevent the separation of suchstructures from occurring. This embodiment may also be used to captureand detect small molecules and bio-molecules.

FIGS. 18 and 19 illustrate the electrophoresis apparatus 10 havingmatrix-like assembly antibodies along the interior surfaces of theseparation capillaries 28, 30, and 32. That is, the affinity 37 elementsmay be also covalently bonded directly to the inner wall of thecapillary or to beads covalently bound to each other and also bound tothe inner wall of the capillary. The use of covalent bonds to bind beadswithin a matrix is also described in U.S. Pat. No. 5,202,010, which isreferred to as beaded capillaries. The attachment of beads to thecapillary through covalent bonds may produce strong bonds that can holdthe beads in the predetermined location along the capillary.

FIG. 20 illustrates the process undertaken to isolate the monovalentantibody fragment Fab′. The antibodies may be obtained by subjectingpurified IgG antibody to two partial enzymatic digestions to obtainF(ab′)2 fragment. The resulting F(ab′)2 antibody fragment may be furtherreduced to produce monovalent Fab′ antibody fragments. As shown in FIG.21, the Fab′ antibody fragment attaches to the inner wall of thecapillary by creating cross-links or bridge chemistries between asulfhydryl group of the antibody fragment Fab′ and an amino group of achemical arm bound to the silanol groups of the inner surface of thefused-silica (quartz) capillary or the surface of beaded structures orpolymeric microstructures having terminal silanol groups. The antibodyfragments attaches to the surface of the separation capillary in anorientation that facilitates the binding of the antibody and the desiredanalyte. A proper orientation of the Fab′ antibody fragments results inan increased surface area of the analyte-concentrator to provide greatercapacity to capture the desired target analyte. A number of antibodiesthat have affinity to a predetermined antigen or hapten may be providedalong a predetermined portion of one or more separation capillaries 28,30, and/or 32. An antigen is a chemical compound that normally causesthe body to produce an antibody when the immunological system in thebody recognizes it. A hapten is a chemical compound that normally doesnot produce an antibody because it is too small and may not berecognized by the immunological system. To produce an antibody for ahapten, the hapten may be bound to an immunogenic carrier (e.g.,albumin, hemocyanin, etc.). This may allow the immunological system torecognize the package (hapten-carrier) as foreign, causing thedevelopment of an antibody. As discussed above, the concentrator 17 mayprovide a number of analytes of interest to the valving system 100through the transport capillary 24. To identify the predetermined numberof analytes of interest, each separation capillary 28, 30, and 32 may beprovided with an antibody that has affinity to a particular analyte. Forexample, as illustrated in FIG. 18, a first type of antibodies 140 thathave affinity to a first analyte provided by the concentrator 17 may beprovided within the interior wall of the separation capillary 28.Likewise, a second type of antibodies 142 and a third type of antibodies144 that have affinity to a second analyte and third analyte may beprovided within the interior walls of the separation capillaries 30 and32, respectively.

FIG. 18 illustrates a valving system 100 that allows the concentratedanalytes from the concentrator 17 to pass through the first, second, andthird antibodies 140, 142, and 144. The transport capillary 24 may bestaggered from one separation capillary to another to form an elongatedanalyte concentrator. For instance, the transport capillary 24 may bestaggered at the separation capillaries 28, 30, and 32 forming elongatedanalyte concentrators 140, 142, and 144. To pass the concentratedanalytes through the valving system 100, the valves 104, 108, 110, 114,116, and 120 along the separation capillaries 28, 30, and 32 may beclosed, and the valves 102, 106, 112, and 118 along the transportcapillary 24 may be opened. Once the output valve 18 is opened, and theanalytes bound to the concentrator 17 are eluted, as described in step107 in FIG. 12, so that the concentrated analytes of interest flowthrough the first, second, and third types of antibodies 140, 142, and144. As such, the antibodies that have affinity to a particular type ofanalyte may bind to that analyte. For example, as the concentratedanalytes pass through the first antibodies 140, the first analytes ofinterest from the concentrated analytes from the concentrator 17 coupleto the first antibodies 140, then as the remaining concentrated analytespass through the second and third antibodies 142 and 144, the second andthird analytes of interest couple to the second and third antibodies,respectively. The remaining concentrated analytes can then be discardedto the waste container 27.

With the desired analytes bound to the antibodies 140, 142, and 144, theconditioning, separating and eluting buffer solution from the cups 40,40′, 40″, 42, 42′, 42″, and 44, 44′, 44″ may be provided to theimmobilized antibodies or antibody fragments, to release and separatethe bound analytes from the immuno complex. This may be accomplished byclosing the valves 102, 106, 112, and 118 along the transport capillary24, and opening the valves 104, 108, 110, 114, 116, and 120 to providethe separation buffer solutions from the cups 40, 42, and 44. For theseparating step, the separating buffer solution may be provided eitherthrough the cups 40, 42, and 44 or through the cups 128, 130, and 132 asdiscussed above in FIG. 17. To capture cells, organelles, and/or otherbulky structures, the concentrator 17 may not be needed.

FIG. 19 also illustrates the addition of valves 152, 154, and 156 tocontrol the flow of buffer solutions in cups 128, 130, and 132 into therespective separation capillaries 28, 30, and 32. The valves 108, 114,and 120 are opened when the capillaries 28, 30, and 32 are filled withconditioning buffer from cups 40, 42, and 44. Then the valves 152, 154,and 156 may be opened to allow the separation buffer from the cups 128,130, 132 to enter into the respective auxiliary capillaries 28, 30, and32. As the electric charge creates an electroosmotic flow in thedirection of the detection zone, the separation buffer entering thecapillaries 28, 30, and 32 downstream from the concentrators flowtowards the detection zone as well. The electroosmotic flow created bythe electricity moves the analytes along the separation buffer towardsthe detection system, allowing separation of the elements to take place.

Having the antibodies within the interior surface of the separationcapillary may provide a larger surface area of antibodies if the lengthof the surface is several centimeters, for example. In other words, moreantibodies may be provided along the longer path that the concentratedanalytes flow through. This means that greater quantity of a particulartype of analyte may be isolated from the concentration of analytes. Inaddition, with the valving system 100, a number of different types ofanalytes in greater quantity may be identified through the differenttypes of antibodies 140, 142, and 144. The diameter of the separationcapillaries may be varied so that large size analytes such as cells,subcellular particles, or globules may pass through the separationcapillaries and couple to the corresponding antibodies. Accordingly, avariety of analytes with a wide range of sizes may be isolated with theantibodies along the inner surface of the capillaries. In addition, theconcentrators may be utilized as a capture matrix to purify at least onetype of analyte present in a simple solution that has reduced number ofchemical and/or biochemical compounds. The concentrator may be alsoutilized to purify at least one analyte from a complex solution that hasgreater number of chemicals and/or biochemical compounds than the simplesolution. With the concentrator, a variety of chemical reactions may beperformed such as multi-component chemical reactions biochemicalreactions, and multi-component biochemical reactions.

The length of the portion of the capillary in which the antibodies arebound along the separation capillary may vary. For example, the antibody140 formed within the separation capillary 28 may be shorten orelongated depending on the quantity of the analytes to be isolated. Forgreater quantity, the length of the antibody formed along the capillary28 may be lengthened.

FIG. 22 illustrates a separation capillary 28 having more than one typeof antibody within its interior wall between the valves 104 and 108. Theseparation capillary 28 may be divided into many portions, where eachportion has one type of antibodies to isolate a particular type ofanalyte. For example, the separation capillary 28 may have differenttypes of antibodies 140, 150, and 160 each having affinity to differenttype of analyte. As such, the separation capillary may isolate a numberof different types of analytes. The separation capillary 28 may beelongated to incorporate more antibodies if desired. The transportcapillary 24 may be coupled to the separation capillary 28 near thevalve 108 to provide the concentrated analytes from the concentrator 17.As the concentrated analytes pass through the separation capillary 28,each of the antibodies may couple to the desired analytes.

A certain antibody may require a different eluting buffer solution tocause that antibody to release the analyte. In such a case, a number ofeluting buffer solutions may be provided through valve 104 so that allof the antibodies release its analyte. After the eluting step, theseparation buffer solution may be provided through the valve 104 aswell. Alternatively, to minimize the adverse affect on the antibodies,the separation buffer solution may be provided down stream from the lastantibodies 160 through the EPC 122 as discussed above. The separatedanalytes are then pass through the detecting zone 45 to identifying theindividual analytes.

The antibody may be any type of affinity interacting chemical orbiological system that attracts a particular analyte. FIGS. 23A and 23Billustrate enlarged views of the antibodies 140, 150, and 160 along theinterior surface of the separation capillary 28. Each antibody generallyhas a shape that is coupled to a substrate, which in this case is theinterior surface of the separation capillary 28. The Y shape antibodyincludes two arms and one stem that imbeds into the substrate. As such,the antibody is immobile, but the two arms have affinity for aparticular analyte (one in each arm) and as that analyte passes acrossthe antibody, the two arms bond to the analyte until the eluting buffersolution interacts with the antibody to release the analyte. Forexample, in FIG. 23A, the two arms for the antibodies 140 have affinityfor the circular analyte but not the square analytes or the triangularanalytes. In contrast, the two branches for the antibodies 160 haveaffinity for the square analyte but not the circular analytes or thetriangular analytes. Other antibodies in the separation capillaries 28,however, may have affinity for the triangular analytes and bond to thetriangular analytes.

FIG. 23B illustrates polymeric microstructures with Y shape antibodyhaving affinity for a particular analyte within the concentrator areawithout the need for frits. Each beaded microstructure may have anantibody that has affinity for a different analyte.

FIGS. 24A and 24B illustrate the use of an antibody like Fab′ asdescribed above. In contrast to the antibodies shown in FIGS. 23A and23B, these Fab′ antibodies have one side of the original antibody. Theantibodies are attached to the substrate by a portion of the originalstem, allowing each group of antibodies to retain its specificity,attracting and bonding to only one type of analyte.

FIGS. 25-27 illustrate a microextraction device 200 having fourtubing-connecting ports: two ports 210 couple to the transport capillary24, and two other ports 214 couple to separation capillary 28, forexample. The two ports 210 for the transport capillary 24 may be largerthan the two ports 214 for the separation capillary to accommodate thelarger size opening in the transport capillary 24. Port 210 may beformed from fused-silica, port 214 may be formed from a plastic tube. Asillustrated in FIG. 27A, the two ports 210 and 214 intersect to form aconcentration area 246. The microextraction device 200 may also have afilling port 252 that provides access to the concentration area 246. Thefilling port 252 may be provided at the central part of themicroextraction device 200. With the filing port 252, prepared by usingcontrolled pore glass (CPG) beads having covalently attached antibodyfragments to their surfaces may be inserted into the concentration area246. This feature allows the coated beads to be replaced as theperformance of the immobilized antibody fragments degrades afterrepeated usage.

The ports 210 and 214 may be formed within the base 202, and the filingport 252 may be formed on the cover 208. The base 202 may have openings230, 232, 234, and 236 that pass through the corresponding ports 210 and214. The openings 230, 232, 234, and 236 may be adapted to receive theelongated portion of valves 218, 220, 222, and 224 that are able to movebetween first and second positions. As illustrated in FIG. 25, eachvalve may have a protruding portion 226 with a cutout 228 to control theflow of fluid through the respective capillary. The cutout 228 may alsobe a hole found through the protruding portion 226. The hole may becoated with glass. To enable normal electrosmotic flow of liquid throughthe cutout 228, the cutout 228 may be formed from fused silica or coatedwith fused silica to maintain a closed connection with the fused-silicacapillaries for the CPG beads.

The port 210 may be substantially aligned with the longitudinaldirection of the separation capillary 28, and the port 214 may besubstantially aligned with the longitudinal direction of the transportcapillary 24. The port 214 may have a larger opening than the openingfor the port 210 to allow greater flow rate of the sample solution fromthe transport capillary 24. Likewise, the transport capillary 24 mayhave a larger opening than the separation capillaries for greater flowrate.

As illustrated in FIG. 25, an indicating arrow 238 may be provided onthe valve so that if the direction of the indicating arrow is in linewith the longitudinal axis of the capillary then the valve is in thefirst position, and if the direction of the indicating arrow isperpendicular to the longitudinal axis of the capillary then the valveis the second position. In the first position, the cut out 228 isaligned with the longitudinal direction of the port to allow the fluidto pass through the port. In the second position, however, the cut out228 faces away from the port so that the protruding portion of the valveblocks the flow of fluid through the port. A connector 240 may beprovided to couple the microextraction device 200 to the transport andseparation capillaries. For instance, in FIG. 26, the connector 240 maybe used to couple the capillary 28 to the port 210 so that the fluidfrom the capillary 28 may be passed to the port 210.

FIG. 26 illustrates a cut out view of the intersection area 246 formedby the intersection of the ports 210 and 214. This way, if the valves218 and 222 are in the first position and the valves 220 and 224 are inthe second position, the fluid from the capillary 28 may pass throughthe port 210, and then to the capillary 28 on the other end of the base202 towards the detection device. Likewise, if the valves 220 and 224are in the first position and the valves 218 and 220 are in the secondposition, the fluid from the capillary 24 may pass through the ports 214and 216, and then to the transport capillary 24 on the other end of thebase 202.

As further illustrated in FIG. 27A, the intersection area 246 may havebulging members 248 along the corners of the channels 210, 212, 214, and216. FIG. 27B is an enlarged view of the intersection area 246illustrated in FIG. 27A. The bulging members 248 along the ports providefor a restricted area in the area 246 such that the gaps through theports in the intersection area are smaller than the size of the beads ormatrix 250, thereby preventing the beads or matrix from moving out ofthe intersection area 246. As such, the beads or the matrix may capturethe desired analytes as the sample passes through the intersection area246.

As illustrated in FIGS. 28A and 28B, the port 210 that is aligned withthe transport capillary may be staggered to form an elongatedconcentration area 246. This allows additional matrix-like assembly orbeads 250 to be incorporated into the concentration area 246 to attractthe desired analyte from the sample solution. In addition, the bulgingmembers 248 may be provided near the intersection area 246 to containthe beads within the intersection area.

FIG. 25 illustrates that the cover 208 may have a filling port 252adapted to receive a cap 254. The filling port 252 may be provided toinsert the beads 250 or matrix into the intersection area 246 to capturethe desired analytes. Once the beads 250 are inserted into theintersection area, the cap 254 may be used to enclose the filling port252. Moreover, the beads 250 may be replaced through a variety ofmethods. For instance, the beads in the intersection area 246 may beremoved by opening the cap 254 so that the beads are exposed through thefilling port 252. The beads may then be removed through a vacuum sourcesuch as a syringe. Once the old beads are removed, a new set of beadswith affinity for a desired analyte may be inserted to the intersectionarea 246 through the filling port 254. To secure the new beads withinthe intersection area, the cap 254 may enclose the filling port 252.

Experimental Data

The following is experimental data for the above invention. Whererelevant, references are cited in the following discussion. A list ofcited references is provided under the subheading “References” in thisspecification. For this experiment, a simple, solid-phase,microextraction device was fabricated for use in on-line, immunoaffinitycapillary electrophoresis. The device, designed in the form of afour-part cross-shaped or cruciform configuration, included a large-boretube to transport samples and washing buffers, and a small-borefused-silica capillary for separation of analytes. At the intersectionof the transport and separation tubes, a small cavity was fabricated,termed the analyte concentrator-microreactor, which contained fourporous walls or semi-permeable membranes (one for each inlet and outletof the tubes) permitting the confinement of beads or suitablemicrostructures. The surface of the beads in the analyte concentratorcarried a molecular recognition adsorbing chemical or affinity ligandsmaterial. The improved cruciform configuration of the analyteconcentrator-microreactor device, designed for use in on-lineimmunoaffinity capillary electrophoresis, enables it to specificallytrap, enrich and elute an analyte from any biological fluid or tissuesample extract without any sample pretreatment except filtration,centrifugation, and/or dilution allowing the separation andcharacterization of target analyte(s) with improved speed, sensitivity,and lower cost than existing techniques.

As a model system, Fab′ fragments derived from a purified IgG antibodywere covalently bound to controlled-porosity glass and used asconstituents of the analyte-microreactor device. The high-specificitypolyclonal antibodies employed in these experiments were individuallyraised against the acidic non-steroidal anti-inflammatory drugsibuprofen and naproxen, and the neuropeptides angiotensin II, andneurotensin. These compounds, which were present in simple and complexmatrices were captured by and eluted from the analyteconcentrator-microreactor using a 50 mM sodium tetraborate buffersolution, pH 9.0, followed by a 100-nL plug of 300 mM glycine buffer, pH3.4, or preferentially a 100-nL plug of 10 mM phosphate-buffered saline,pH 7.4, containing 20-50% acetonitrile. Two analyte concentrators weretested independently: one containing Fab′ fragments derived fromantibodies raised against ibuprofen and naproxen; the other containingFab′ fragments derived from antibodies raised against angiotensin II andneurotensin. Each resulting electropherogram demonstrated the presenceof two eluted materials in less than 20 min.

Immunoaffinity capillary electrophoresis performed in a cruciformstructure was simpler and faster than previously reported in theliterature using on-line microextraction devices designed in a linearformat. The new concentration-separation system operated consistentlyfor many runs, maintaining reproducible migration times and peak areasfor every analyte studied.

This microextraction device design has been fabricated for facilitatingthe rapid introduction of samples and cleaning buffers through alarge-bore transport tube, and for improving the determination ofaffinity-bound target analytes employing a small-bore separationcapillary, maintained free of contamination, after multiple uses. Theon-line extraction approach using immunoaffinity capillaryelectrophoresis is illustrated by determining the acidic drugs ibuprofenand naproxen, and the peptides angiotensin II and neurotensin in urineat concentration levels of less than 5 ng/mL, when the separatedanalytes were monitored at 214 nm. Furthermore, the microstructureswithin the cavity of the analyte concentrator-microreactor containingsuitable immobilized antibodies were re-used several times, and whentheir performance diminished, it was possible to readily replace themwith new ones.

Materials and Methods

Chemicals:

All chemicals were of the highest quality reagent grade. Deionized,double-distilled water was purified with a MILLI-Q® Plus Ultra-Purewater system from Millipore Corporation (Bedford, Mass., USA). Nylonfilters (0.20 μm) used to remove particulate matter were obtained fromGelman Sciences (Ann Arbor, Mich., USA). Underivatized controlled poreglass (CPG) beads (3000 Å pore size, 200-400 mesh, irregularly shaped)were purchased from CPG Inc. (Fairfield, N.J., USA). Bare fused-silicacapillary columns were obtained from Polymicro Technologies (Phoenix,Ariz., USA). Sulfosuccinimidyl 4-(N-maleidomethyl)cyclohexane-1-carboxylate (SSMCC), the immunoPure F(ab′)2 preparationkit, 2-mercaptoethylamine.HCl, BLUE CARRIER® immunogenic protein, andpepsin agarose were purchased from Pierce Biotechnology (Rockford, Ill.,USA). 3-Aminopropyl-triethoxysilane was obtained from Polysciences(Warrington, Pa., USA). S-(+)-Ibuprofen((S)-(+)-2-(4-isobutylphenyl)propionic acid), S-(+)-naproxen((S)-(+)-2-(6-methoxy-2-naphthyl)propionic acid), phenylmethylsulfonylfluoride, soybean trypsin inhibitor, iodoacetate,p-aminobenzamidine.HCl, leupeptin hydrochloride, potassium chloride,sodium phosphate (Na2PO4), and potassium phosphate (KH2PO4) werepurchased from Sigma-Aldrich (St. Louis, Mo., USA).Peptide-N-glycosidase F (PNGase F) was obtained from New England Biolabs(Beverly, Mass., USA). Sodium thiocyanate and sodium azide werepurchased from-Fisher Scientific (Pittsburgh, Pa., USA). Superdex-75resin, PD-10 desalting column, and r-Protein A SEPHAROSE® were purchasedfrom Amersham Pharmacia Biotech (Piscataway, N.J., USA). Angiotensin Hand neurotensin were obtained from Peninsula Laboratories (Belmont,Calif., USA) and Sigma-Aldrich. Methanol was purchased from AlliedSignal, Burdick & Jackson (Muskegon, Mich., USA). SEP-PAK® C18cartridges were obtained from Waters Corporation (Milford, Mass., USA).A concentrated (10-fold) phosphate-buffered saline solution was preparedas follows: dissolved 80 g of NaCl, 2.0 g of KCl, 14.4 g of Na2HPO4, and2.4 g of KH2PO4 in 800 ml deionized, double-distilled water; adjust thepH to 7.40 with HCl; adjusted the volume to 1 liter with additionaldeionized, double-distilled water; filtered with Nylon filters (0.20μm). The final concentration of the used phosphate-buffered saline (a1:10 dilution from the concentrated) is approximately the following:0.010 M sodium phosphate (dibasic) buffer, pH 7.40, containing 0.0027 Mpotassium chloride, 0.137 M sodium chloride, and 0.0018 M potassiumphosphate (monobasic).

Fresh, stock solutions of the peptides dissolved in water at aconcentration of 100 μg/nL were prepared prior to use. The acidic drugsibuprofen and naproxen were dissolved in a methanolic solution (35:65methanol:water, v/v), also at a concentration of 100 μg/mL.

Methods:

Preparation of Urine Samples.

Urine samples from healthy males were collected as morning clean-catchurine specimens, prior to breakfast and with a simple dinner theprevious evening. Immediately after collection of a pool of six urinesamples, a cocktail of protease inhibitors was added including 0.1 mM ofPMSF, soybean trypsin inhibitor, iodoacetate, p-aminobenzamidine, and 1mM leupeptin [1,2,3]. A 0.20-μm porous diameter Nylon filter was used tofilter the pooled samples, to remove particulate matter or cells.Diluted and undiluted urine samples were used for the experiments.Undiluted urine specimens were spiked with the analytes prior toOn-line, immunoaffinity capillary electrophoresis (IACE) by adding thetwo acidic drugs into one aliquot of urine sample, and the two peptidesinto a second aliquot of urine sample, with final concentrations of 1-,2-, 5-, and 50-ng/mL respectively. Conversely, urine samples werediluted first (1:1 and 1:5, v/v) with 50 mM sodium tetraborate buffer,pH 9.0, and then spiked with the analytes, as described above, at thesame final concentrations as the undiluted samples.

Preparation of Antibodies.

Polyclonal antibodies raised against commercially available ibuprofen,naproxen, angiotensin II, and neurotensin (FIGS. 29A and 29B), coupledcovalently to BLUE CARRIER® immunogenic protein, were raised in rabbitsusing a method similar to those described elsewhere [3,4]. Thepurification of the antibodies from rabbit antisera was performed byHPLC using r-Protein A affinity chromatography as previously described[4]. Conjugates for immunization were prepared by various methods withthe same modifications [1,5-7]. The method described by Grafe andHoffmann [5] was used to link ibuprofen to the BLUE CARRIER® immunogenicprotein. The method described by Shi et al. [6] was used to linknaprofen to the immunogenic protein. The linking of the peptides to theimmunogenic protein was performed as previously reported for othercarriers [1,7].

The antibodies purified from the antisera by r-Protein A SEPHAROSE®affinity chromatography, were further purified by immunoadsorption on aCPG column containing immobilized haptens (ibuprofen, naproxen,angiotensin II, or neurotensin). In order to accomplish this task, thehaptens ibuprofen and naproxen were covalently linked tocontrolled-porous glass, employing the same chemistries used to link thehaptens to the BLUE CARRIER® immunogenic protein. The peptides werelinked to CPG through a procedure described elsewhere [7]. The columnswere individually eluted with 3 M sodium thiocyanate in 0.01 M sodiumphosphate buffer pH 7.0 [3]. The highly specific, pure antibodies weredialyzed against 0.01 M sodium phosphate buffer pH 7.0, aliquoted insmall fractions, and stored at −70° C. until use.

Polyclonal antibodies raised against commercially available ibuprofen,naproxen, angiotensin II, and neurotensin, coupled covalently to BlueCarrier immunogenic protein, were raised in rabbits using a methodsimilar to those described elsewhere [3,4]. The molecular structures ofibuprofen and naproxen are illustrated in FIG. 29. Angiotensin II andneurotensin are peptide sequences well known to one of skill in the art.The purification of the antibodies from rabbit antisera was performed byHPLC using r-Protein A affinity chromatography as previously described[4]. Conjugates for immunization were prepared by various methods withthe same modifications [1,5-7]. The method described by Grafe andHoffmann [5] was used to link ibuprofen to the Blue Carrier immunogenicprotein. The method described by Shi et al. [6] was used to linknaprofen to the immunogenic protein. The linking of the peptides to theimmunogenic protein was performed as previously reported for othercarriers [1,7].

Immunoadsorbed purified antibodies were subjected to two partialenzymatic digestions to generate F(ab′)2 antibody fragments. The firstdigestion, a deglycosylation process described by Wilson et al. [8], wasperformed to remove N-linked glycosyl groups attached to the Fc fragmentof IgG. These investigators used PNGase F, an enzyme that removesN-linked oligosaccharides. Approximately 20 U/μL PNGase F were incubatedwith 1 mg/mL of purified IgG for 24 hr at 37° C. (FIG. 20).

The second enzymatic digestion was carried out using pepsin to removethe Fc fragment of the IgG while maintaining the intra- andinter-disulfide bridges. This enzymatic process was readily achieved,since the Fc fragment was free of sugars. The removal of some sterichindrance from neighboring carbohydrate moieties near the hinge regionfacilitated the action of pepsin. Pepsinolysis was carried out using acombination of the method described by Wilson et al. [8], and themanufacturer's instructions described in the ImmunoPure F(ab′)2preparation. The divalent F(ab′)2 antibody fragments formed were thenreduced to monovalent Fab′ antibody fragments, by incubation with equalvolumes of 200 mM mercaptoethylamine.HCl reagent for 30 min at 37° C.This step, reported by Phillips and Smith [2], replaced the F(ab′)2reduction with Cleland's reagent (as previously described) because thelatter agent was found to require optimization conditions and it is orcan be an unpredictable reducing agent (FIG. 16).

Coupling of Fab′ Fragments to Glass Beads

Controlled-porous glass beads, previously utilized to link antibodiesdirected against methamphetamine [7,9], were employed to bind monovalentFab′ fragments purified from antibodies raised against the two acidicdrugs and the two neuropeptides [9,10,1,7,11]. The irregularly shapedbeads were incubated at 95° C. for sixty minutes in the presence of 10%aqueous 3-aminopropyltriethoxysilane. This treatment was repeated fourtimes. The incubation was carried out with the beads and solution insidea double side arm glass container, in a temperature-controlled waterbath, with gentle agitation. The access ports of the glass container,inlet and outlet, were sealed with multi-hole plastic caps to reduce theevaporation of the silane solution. The beads were then incubated at 95°C. for sixty minutes with 10 mM hydrochloric acid. The beads were washedwith copious amounts of distilled/deionized water before preparing themaleimide-activated surface. The beads were then incubated at 30° C. forsixty minutes with a buffer solution containing 50 mM sodium borate, pH7.6, and 1 mg/mL SSMCC. The beads were finally washed thoroughly with 50mM sodium borate buffer, pH 7.6, and then incubated overnight at 4° C.with approximately 500 μg/mL of SH-containing Fab′ peptide in 50 mMsodium borate buffer, pH 7.6. The entire process to link SH-containingFab′ fragments to the wall of the capillary is summarized in FIG. 21.

Fabrication of the analyte concentrator-microreactor

The analyte concentrator-microreactor device, designed in a cruciformconfiguration with four entrance-exit ports (FIG. 11A), was made of atransparent acrylic substrate, but other plastic materials (e.g.,TEFLON® fluoropolymer resins, nylon, polyimide, or PEEK® plastic) mayalso be used. Two chromatographic fittings were used to connect twolarge-bore plastic tubes (one inlet, one outlet) to the microextractiondevice to transport sample and washing buffers. Two nanovolume fittings(nomenclature used to describe a device or sleeve to provide a tightfitting to a fused-silica capillary) were used to connect two 100-μmi.d., 360-μm o.d. fused-silica capillaries (one short inlet, one longoutlet) to the device (FIGS. 25-27). Each port contained a porouspolymeric frit, or a constricted area, fabricated from fine pieces ofmaterial produced by a blade from a frit taken from a commerciallyavailable SEP-PAK® C18 cartridge. The semi-permeable frit structurepermitted the confinement of the irregularly shaped CPG beads containingimmobilized Fab′ fragments of IgG within the analyteconcentrator-microreactor. The cavity was filled with CPG beads throughan additional port (termed the filling port) after the appropriatetubing (two PEEK® plastic tubes and two fused-silica capillary tubes) atthe four ports were previously installed. After the cavity was properlyfilled with the coated beads employing a low vacuum aspiration systemand gentle shaking of the device, the filling port that facilitated theentrance of the beads into the system was closed very tightly to preventair from entering the analyte concentrator-microreactor. Since theanalyte concentrator-microreactor device was made of a transparentacrylic substrate, the entire packing process was monitored using astereo microscope.

The solid-phase microextraction device was designed with fourmicrovalves as indicated by circles with cross areas in FIG. 11A and inmore detail in FIGS. 25-27. The micro-fabricated valves permitted fullcontrol of the path of fluid in the appropriate direction, allowing theinteraction of the constituents of the sample under study with theantibody fragments present in the analyte concentrator-microreactordevice.

Once the analyte concentrator-microreactor was completed packed andproperly assembled, one of the inlet positions for the large-bore tubingwas attached by a commercially available connector to a 3-mL plasticsyringe as depicted in FIGS. 30 and 31.

Separation and Detection of Analytes by CE-UV

Capillary electrophoresis studies for ibuprofen and naproxen [12], andangiotensin II and neurotensin [13,14] have been reported previouslyusing commercially available instruments, but not using the cross-shapedanalyte concentrator-microreactor configuration. For experimentsdirected to this application, a capillary electrophoresis apparatus asdepicted in FIG. 31 was employed [4,14]. The fused-silica capillary(100-μm×65-cm×100-cm) used for analyte separation was conditioned priorto being connected to the microextraction device, by rinsing with waterfor two minutes, 0.5 N NaOH for 5 min, water for three minutes, and withbackground electrolyte (as specified below) for five minutes. Theintroduction of sample and washing buffer was achieved by using theappropriate valves to direct the flow of liquid (FIGS. 11A and 25-27).When the valves for the transport tube were closed, the separationcapillary was conditioned with 50 mM sodium tetraborate, pH 8.5. Whenthe valves for the separation capillary were closed, samples wereintroduced into the analyte concentrator-microreactor using a large-boreplastic tube by positive pressure using a syringe, or by employing a lowvacuum aspiration system directly from the sample reservoir. The amountof sample introduced into the microextraction device was approximately 1mL, a sufficient volume containing enough concentration of hapten tosaturate the binding sites of the antibody fragments. Standards wereprepared in 50 mM sodium tetraborate buffer, pH 9.0. Spiked urinesamples were prepared by adding the analytes directly to undilutedurine, or to diluted urine in 50 mM sodium tetraborate buffer, pH 9.0.The sample was allowed to be in direct contact with the immobilizedaffinity ligand for five minutes, permitting the peptide to be retainedin order to control the appropriate interaction and temperature forachieving maximum binding. After a few washes of the transport tube with50 mM sodium tetraborate buffer, pH 9.0, the valves were switched to theseparation position. The separation column was conditioned once morewith 50 mM sodium tetraborate buffer pH 8.5, and completely degassed.The peptide was finally eluted with a plug of approximately 100 nL of300 mM glycine-HCl buffer, pH 3.4, or preferentially 100 nL of 10 mMphosphate-buffered saline, pH 7.4, containing 20-50% (v/v) acetonitrile,and the separation was allowed to continue. (The glycine buffer is usednormally when the peptides are labeled with a fluorescence chromophore,and fluorescence detection is employed. The phosphate-buffered saline,containing acetonitrile or other organic solvent, is normally used whenthe peptides are not tagged by a fluorescence chromophore, andultraviolet detection is employed.)

All separations were performed at 26 kV, and the majority of thecapillary was positioned within a cartridge cassette [15,4,14],containing a fluid of regulated-temperature, in order to maintain thecapillary temperature at approximately 25oC. The separated analytes weremonitored by UV-absorption preferentially at 214 nm (but a wide range ofwavelengths can be used as well), using a modified on-column UV,variable-wavelength detection system (Hitachi Instruments, Inc.,Danbury, Conn., USA). Data collection for quantification of theelectropherographic peaks was carried out with a Chromato-Integrator(Hitachi Instruments, Inc.).

When not in use, all separation capillaries were removed from themicroextraction device and rinsed with sequential washes of water, 0.1 NNaOH, water, 0.5 N HCl, and water for approximately 5 min each, and thenstored in air at 25oC. The area of the microextraction device wasmaintained wet at all times with a solution of 50 mM sodium tetraboratebuffer, pH 7.0, containing 1% (w/v) sodium azide. The microfabricatedvalves were kept closed to allow the tetraborate-azide buffer to remainwithin the microextraction device. The disassembled microextrationdevice was stored at 4oC.

Results and Discussion.

Improved Procedure to Obtain Fab′ Fragments. (FIG. 20.)

One of the standard procedures to obtain Fab′ fragments containing thehinge region cysteine(s) is to generate first F(ab′)2 fragments.Traditionally, pepsin has been the preferred proteolytic enzyme tocleave the Fc fragment of several IgGs, including IgY [56], whencompared to bromelain, ficin, and lysyl endopeptidase [16,17]. However,a wide range of optimization conditions for the cleavage of the Fcfragment constituent of the monomeric IgG, have been reported in theliterature [17-20]. Apparently, the different subclasses of IgGs presenta certain degree of resistance to pepsin cleavage producing a widevariation in the yield for the formation of F(ab′)2 fragments, dependingof the IgG subclass. This is due, in part, by investigators whounderestimated the steric effect on the active site of pepsin, which canbe attributed to the presence of N-linked carbohydrates near the hingeof IgG. Wilson et al. [8], demonstrated that by removing thecarbohydrate groups of the intact IgG molecule, prior to digestion bypepsin, the yield for the formation of F(ab′)2 fragments increaseddramatically. The presence of carbohydrates in antibodies seems to beimportant for antigen clearance functions, such as complementactivation, and antibody activity [21]

In the experiments reported here, it was confirmed that the removal ofN-linked carbohydrates was preferred for facilitating pepsin activity onthe IgG molecule, to generate dimeric F(ab′)2 fragments and thenmonomeric Fab′ fragments (see FIG. 20). Comparative pepsinolysis studiesof IgG, with and without glycosylation, demonstrated a more consistentyield of the monomeric deglycosylated IgG when qualitative andquantitative capillary electrophoresis studies were performed (data notshown). Covalent attachment of monomeric Fab′ fragments throughSH-groups permits proper orientation of the molecule and increases thesurface area to enhance the capturing of a target ligand. Specificallyoriented attachments of antibodies, or antibody fragments, to a surfacehave been demonstrated to be more efficient in capturing a targetanalyte, when compared to chemistries employing random attachments[22-24]. The advantages of oriented immobilization of biologicallyactive proteins are: (a) improved steric accessibility to the activebinding sites; (b) increased stability of the immobilized molecule; and(c) facilitation to a greater surface area of affinity interaction.

Determination of Pharmaceutical Drugs and Peptides in Urine Specimens

Determination of non-steroidal anti-inflammatory drugs and neuropeptidesin urine was carried out by immunoaffinity capillary electrophoresis.Several experiments were performed to test the efficiency of the system.In a preliminary study, a 50-ng/mL solution of angiotensin II wasapplied to the analyte concentrator-microreactor device containingimmobilized Fab′ fragments derived from a polyclonal antibody raisedagainst the peptide. The device was part of the capillaryelectrophoresis instrument depicted in FIG. 31. The sample was allowedto be in direct contact with the immobilized affinity ligand, thepeptide was retained, and after a few washes and conditioning with theappropriate buffers, the peptide was eluted with a small plug of 0.3 Mglycine-HCl buffer, pH 3.4, or neutral pH buffers containingacetonitrile, such as phosphate-buffered saline, pH 7.4, containing20-50% acetonitrile, or other organic solvents at concentrations rangingfrom 5% to 100% (apparently, there is not a universal way of eluting allantibodies or antibody fragments. It is preferred to optimize everyelution condition for each individual immobilized affinity ligand-targetanalyte complex. Since the affinity binding is different for everyimmunological complex, it may affect the linearity of quantification ifthe hapten is not released completely from the immunological complex).The success of this experiment prompted optimization of the bindingconditions. A series of dilutions were performed in the urine specimen,and spiked with the acidic drugs or peptides. A representativeelectropherogram is shown in FIGS. 32A and 32B. The urine specimen usedin this experiment was diluted 1:1 (v/v) with 50 mM sodium tetraboratebuffer, pH 9.0. The sample was divided in two aliquots. One aliquot wasspiked with 5 ng/mL each of ibuprofen and naproxen, and another with 5ng/mL each of angiotensin II and neurotensin. The samples were analyzedin two separate analyte concentrator-microreactors. The data obtainedwas consistent for nine runs, maintaining reproducible migration timesand peak areas as indicated in Table 1 below. Sample Ibuprofen NaproxenAngiotensin II Neurotensin Control sample 14.40* 17.20* 13.30* 16.40* (5ng/mL)  1845+  1470+   955+  1580+ (dilution 1:1) Same as control 14.47*17.25* 13.35* 16.44* (Assay No. 9)  1837+  1463+   950+  1568+ Same ascontrol 14.50* 17.31* 13.39* 16.48* (after 3 month at  1657+  1324+  848+  1417+ 40° C. and with azide) Same as control 14.48* 17.26*13.34* 16.45* (dilution 1:5)  1893+  1486+   975+  1598+ Sample at14.46* 17.28* 13.37* 16.47* 2 ng/mL   718+   564+   385+   637+(dilution 1:1) Sample at 14.43* 17.24* 13.35* 16.44* 1 ng/mL   293+  185+   193+   321+ (dilution at 1:1) Sample at 14.38* 17.12* 13.17*16.34* 5 ng/mL  1417+  1215+   821+  1411+ (undiluted) Sample at 14.36*17.09* 13.11* 16.29* 1 ng/mL   248+   163+   185+   314+ (undiluted)Note that the numbers with (*) represent migration time in minutes; andnumbers with (+) represent peak area in arbitrary units.

The analyte concentrator-microreactor needs to be packed correctly.Otherwise, incorrectly filling of the device can lead to discontinuityin the current. The filled device was examined with the assistance of astereo microscope to monitor packing efficiency. After three months at40 C and in the presence of sodium azide, the binding activity for mostantibody fragments was still maintained at approximately 90% activity(Table 1).

Maintaining the appropriate pH was also useful, as demonstrated in thecomparative studies performed for the diluted and undiluted urinespecimens. As seen in Table 1, undiluted urines yield peak areas muchlower than diluted urines, while giving very similar migration times. Ithas been known for many years, specificity is the ability of antibodiesto discriminate among different ligands. In the case of haptens,extremely fine structural changes in the molecule are responsible forthe discrimination [25]. To maintain the so called exquisitespecificity, the molecular recognition properties needs to be kept atoptimum conditions to be effective. Ionic strength and pH of the bufferis another factor in maximizing the binding. Since urine samples havedifferent pHs, which is dependent on many factors, it would beappropriate to bring the pH of each urine tested to a value of 9.0, butthis may be impractical. A dilution of 1:1 (v/v) with 50 mM sodiumtetraborate buffer seems to be much more convenient and consistent withthe needs of an automated system.

Higher dilutions of urine specimens, e.g., 1:5 (v/v), with 50 mM sodiumtetraborate buffer, pH 9.0, provides improved replicates for peak areaswhen compared to the 1:1 dilutions, and thus confirms the importance ofpH. The improved analyte concentrator-microreactor structure describedin this paper has the following attractive features: (a) it usesmicroliter sample volumes; (b) extensive sample preparation is notrequired, except filtration, centrifugation, and/or dilution; (c)protects the separation capillary from non-specific binding of unwantedmaterials, because of its cruciform design; (d) operates consistentlyfor many runs; (e) yields reproducible migration times and peak areasfor the analyte under study; (f) permits easy replacement of CPG beads;(g) allows the on-line concentration of samples to increase up to1000-fold or more, thereby permitting quantitation levels of analytes atapproximately 5 ng/mL or lower using UV detection.

FIGS. 33A, 33B, and 33C illustrate electropherogram for analytesdetected using the electrophoresis apparatus 10 with three concentratorswith a different antibody in each of the concentrators. In this case,three antibodies of Fab′ fragments which act against the followingpeptide hormones were used: (A) neurotensin; (B) enkephalin; and (C)cholecytokinin. The concentrators 34, 36, and 38, each had an antibodywith affinity towards (A) neurotensin; (B) enkephalin; and (C)cholecytokinin, respectively. Urine specimen were spiked with theneurotensin, enkephalin, and cholecytokinin analytes prior to IACE. Theurine specimen were then pass through the transport capillary 24 towardsthe three concentrators 34, 36, and 38. After the three antibodiescaptured their respective analytes, the separation capillaries 28, 30,and 32 were eluted sequentially. FIG. 33 indicates that migration timeand peak for the three analytes: (A) neurotensin; (B) enkephalin; and(C) cholecytokinin correspond the control sample illustrating that theelectrophoresis apparatus 10 operates consistently for the analytesunder study. The process of capturing each peptide from the urinespecimen by immobilized antibody fragments located at the analyteconcentrator, cleaning the capillary with an appropriate buffer, elutingthe bound peptide from the immobilized affinity ligand, and separatingthe released peptide by capillary electrophoresis was carried under thesame experimental conditions as described above in the ExperimentalData.

FIG. 34 illustrates a diagnostic kit 260 that may be used by individualsto detect early signs of certain disease(s). Some individuals may bepredisposed to certain diseases more so than others based on theirfamily health history, such as cancer, diabetes, and heart diseases. Forthese individuals, an early detection of such diseases may be a key tofighting the diseases. In this regard, individuals may use thediagnostic kit 260 to monitor and detect early signs of a number ofdiseases. Such tests may be done at the home of the individual forconvenience and privacy. The diagnostic kit 260 may include theelectrophoresis apparatus 10 that is communicatably coupled to a CPU 262that may operate the electrophoresis apparatus 10 based on apredetermined set of instructions. As discussed above, the valves on thetransport and separation capillaries may be motor operated, which arecontrolled by the CPU.

An individual who is predisposed to a predetermined disease may selector purchase a system of capillaries and valves with the concentrators34, 36, and 38 that may isolate biomarkers that are associated with apredetermined disease. In general, each disease may have a plurality ofbiomarkers or analytes associated with that disease. Different diseasesmay have different biomarkers than other diseases. As such, biomarkersmay serve as a fingerprint for identifying a particular disease anindividual may have based on test performed on the individual'sspecimen. If the biomarkers are detected, then evaluation may be made asto whether the biomarkers correspond to a particular disease or not. Forinstance, Disease 1 may be associated with four biomarkers: A, K, M, andT; Disease 2 may be associated with five biomarkers: B, D, F, L, and P;and Disease 3 may be associated with three biomarkers: B, T, and Y. Eachbiomarker may have its migration time through the separation capillaryand peak that may be detected by the detector 86. If an individual ispredisposed or concerned about disease 2, then the individual may selecta system of capillaries and valves with at least five analyteconcentrators where each analyte concentrator has an affinity towardsthe analytes or biomarkers B, D, F, L, and P, respectively, or in anyorder. In the case of detecting disease 3 with three biomarkers,concentrators 34, 36, and 38 as illustrated in FIG. 9 or FIG. 14 may beused to isolate biomarkers B, T, and Y, respectively. Alternatively, asillustrated in FIG. 22, a separation capillary 28 having three types ofantibodies 140, 150, and 160 within its interior wall between the valves104 and 108 may be used to isolate the biomarkers B, T, and Y, in anyorder. Likewise, the separation capillaries 30 and 32 may be used toisolate biomarkers A, K, M, and T for disease 1, and biomarkers B, D, F,L, and P for disease 2, respectively. As such, one system of capillariesand valves may be used to isolate biomarkers for more than one disease.

The individual may install the system of capillaries and valves into theplatform 12 and locked it in placed with the holders 49. For isolatingthe biomarkers, the individual's specimen such as urine may be providedinto the sample cup 15. Other specimens such as blood, hair, and nailmay be provided. The CPU may then send the control signals 266 tooperate the apparatus 10 according to the steps generally discussed inFIG. 12 to isolate the analytes of interest or the biomarkers from thespecimen provided by the individual. The detector 86 may then obtain thedata for each of the biomarkers in terms of their respective migrationtimes and peaks. For instance, if the individual providing the specimendoes have the disease 3, then the detector 86 may find three biomarkersB, T, and Y, each having its respective migration time through theseparation capillary and peak. On the other hand, if the individual doesnot have the disease 3, then one or two of the biomarkers may bedetected from the specimen but not all three biomarkers. This datainformation 268 may be analyzed in a variety of ways. For instance, thedata information 268 may be provided to the CPU 262, which is thencompared with the plurality of reference data stored in the memory 264.The CPU may find that the biomarkers do indicate that the specimenprovided by the individual has the disease 2 if all three biomarkers arefound to have substantially similar respective migration times and peaksas compared to the migration times and peaks indicated in the referencedata stored in the memory 264. On the other hand, if at least one of thebiomarkers do not substantially match up with the migration time and thepeak, then the CPU may indicate that the individual may not have disease2.

To check on the test result from the CPU, the individual may send thedata 268 to an evaluator 270 such as a specialist or doctor to examinethe data to confirm or deny that the biomarkers correspond to a disease.The evaluator may provide a feedback 272 to the CPU 262 so that theindividual may take the next step based on the feedback provided by theevaluator. The memory may be updated by the evaluator if new biomarkersare found that corresponds to a particular disease. In addition, theevaluator 270 and the memory 264 may be provided remotely and the data268 and feedback 272 may be provided electronically such as through theInternet. Alternatively, the CPU 262 may send the data information 268directly to the evaluator 270 for analysis of the data and a feedback tothe CPU. In other words, the CPU may skip the comparison of the data 268with the reference data stored in the memory 264 and go directly to theevaluator 270 for the analysis.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein. By their citation of variousreferences in this document, the applicant do not admit that anyparticular reference is “prior art” to his invention. While specificexamples have been provided, the above description is illustrative andnot restrictive. Any one or more of the features and not restrictive.Any one or more of the features of the previously described embodimentscan be combined in any manner with one or more features of any otherembodiments in the present invention. Furthermore, many variations ofthe invention will become apparent to those skilled in the art uponreview of the specification. The scope of the invention should,therefore, be determined not with the reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

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1-321. (canceled)
 322. A controlled electrophoresis method, comprising:introducing a sample into a transport passage of an electrophoresisapparatus; the electrophoresis apparatus including a first separationpassage intersecting the transport passage at a first overlappingportion, a first analyte concentrator at the first overlapping portionto concentrate a first analyte of interest, a second separation passageintersecting the transport passage at a second overlapping portion, anda second analyte concentrator at the second overlapping portion toconcentrate a second analyte of interest; passing the introduced samplein the transport passage into the first overlapping portion so that thefirst analyte of interest in the sample is concentrated in the firstanalyte concentrator; localizing the first analyte concentrator andthereby increasing the concentration of the first analyte of interest bythe first analyte concentrator; passing the introduced sample in thetransport passage into the second overlapping portion so that the secondanalyte of interest in the sample is concentrated in the second analyteconcentrator; localizing the second analyte concentrator and therebyincreasing the concentration of the second analyte of interest by thesecond analyte concentrator; releasing the first analyte of interestconcentrated in the first analyte concentrator into the first separationpassage; releasing the second analyte of interest concentrated in thesecond analyte concentrator into the second separation passage;delivering the released first analyte of interest in the firstseparation passage to a detector system which identifies andcharacterizes the first analyte of interest; and delivering the releasedsecond analyte of interest in the second separation passage to thedetector system which also identifies and characterizes the secondanalyte of interest.
 323. The method of claim 322 wherein the localizingthe first analyte concentrator is after the passing into the firstoverlapping portion and the localizing the second analyte concentratoris after the passing into the second overlapping portion.
 324. Themethod of claim 322 wherein the localizing the first analyteconcentrator includes closing valves associated with the firstoverlapping portion.
 325. The method of claim 322 wherein the localizingthe second analyte concentrator includes closing valves associated withthe second overlapping portion.
 326. The method of claim 322 wherein thelocalizing the first analyte concentrator includes heating same. 327.The method of claim 322 further comprising before the releasing thefirst analyte of interest, subjecting the first analyte concentrator toat least one of gentle shaking, microwave pulsing or acoustic mixing.328. The method of claim 322 wherein the releasing the first analyte ofinterest includes introducing an eluting solution into the firstseparation passage upstream of the first analyte concentrator.
 329. Acontrolled electrophoresis method, comprising: introducing a sample intoa transport passage; passing the introduced sample past a first portionof the transport passage which intersects with an overlapping firstseparation passage so that a first analyte of interest in the sample iscaptured by a first analyte concentrator in the first portion;controlling the temperature of the first analyte concentrator to a firsttemperature associated with the first analyte concentrator; passing theintroduced sample past a second portion of the transport passage whichintersects with an overlapping second separation passage so that asecond analyte of interest in the sample is captured by a second analyteconcentrator in the second portion; controlling the temperature of thesecond analyte concentrator to a different second temperature associatedwith the second analyte concentrator; releasing the captured firstanalyte from the first analyte concentrator into the first separationpassage; causing the released first analyte to pass by electrophoresismigration or by a combination of electrophoresis migration and pressureto a detector system which identifies and characterizes the firstanalyte; releasing the captured second analyte from the second analyteconcentrator into the second separation passage; and causing thereleased second analyte to pass by electrophoresis migration or by acombination of electrophoresis migration and pressure to the detectorsystem which also identifies and characterizes the second analyte ofinterest.
 330. The method of claim 329 wherein the controlling thetemperature of the first analyte concentrator includes closing valvessurrounding the first analyte concentrator.
 331. The method of claim 329wherein the first and second temperatures differ by about 12 degreesCentigrade.
 332. The method of claim 329 wherein one of the first andsecond temperatures is approximately 25 degrees Centigrade and the otheris approximately 37 degrees Centigrade.
 333. The method of claim 329further comprising before the releasing the captured first analyte,subjecting the first analyte concentrator to gentle shaking.
 334. Themethod of claim 329 further comprising before the releasing the capturedfirst analyte, subjecting the first analyte concentrator to microwavepulsing.
 335. The method of claim 329 further comprising before thereleasing the captured first analyte, subjecting the first analyteconcentrator to acoustic mixing.
 336. The method of claim 329 whereinthe releasing the captured first analyte includes introducing an elutingsolution into the first separation passage.
 337. The method of claim 329wherein the controlling the temperature of the first analyteconcentrator includes isolating the first analyte concentrator byclosing valves associated therewith.